Lecture Notes on Translation and DNA Repair

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February 5, 2025

Introduction

In the preceding lecture, we examined the initiation phase of translation, emphasizing the distinctions between prokaryotic and eukaryotic systems. This lecture will conclude our exploration of translation by detailing the elongation and termination phases. We will note the conserved nature of the elongation process across prokaryotes and eukaryotes. Subsequently, we will introduce the topic of DNA repair and genomic stability, laying the groundwork for future discussions in this area.

Elongation Phase

Overview of Elongation

The elongation phase of translation is characterized by the sequential addition of amino acids to a growing polypeptide chain. This process is highly conserved between prokaryotic and eukaryotic organisms, indicating its fundamental importance in cellular life.

Key Components in Elongation

The elongation phase necessitates the coordinated function of several components:

Ribosomes: Serve as the central machinery, providing the structural framework and catalytic activity for peptide bond formation.
Aminoacyl-tRNAs: Act as carriers of amino acids, delivering them to the ribosome according to the mRNA sequence.
Elongation Factors (EFs): Accessory proteins that facilitate and regulate the various steps of elongation, enhancing both the rate and accuracy of protein synthesis.

Elongation Factors: Types and Function

Elongation factors are crucial for the efficient and accurate progression of the elongation phase. They ensure correct aminoacyl-tRNA delivery, utilize GTP hydrolysis for energy and proofreading, and facilitate ribosome translocation.

Prokaryotic Elongation Factors

Prokaryotes utilize two primary elongation factors: EF-Tu and EF-Ts.

EF-Tu

EF-Tu (Elongation Factor Thermo unstable) is responsible for the delivery of aminoacyl-tRNA to the ribosomal A-site. This delivery is facilitated by the formation of a ternary complex consisting of EF-Tu, GTP, and aminoacyl-tRNA. GTP hydrolysis by EF-Tu is essential for its function and for proofreading during codon recognition.

EF-Ts

EF-Ts (Elongation Factor Thermo stable) functions as a guanine nucleotide exchange factor (GEF). It recycles EF-Tu by catalyzing the exchange of GDP for GTP, thus regenerating the active EF-Tu-GTP form required for subsequent elongation cycles.

Eukaryotic Elongation Factors

Eukaryotes employ analogous elongation factors to their prokaryotic counterparts.

EF-1$\alpha$

EF-1$\alpha$ is the eukaryotic functional ortholog of EF-Tu. It performs the same role of delivering aminoacyl-tRNA to the ribosome A-site as a ternary complex with GTP and aminoacyl-tRNA, coupled with GTP hydrolysis.

EF-1$\beta\gamma$

EF-1$\beta\gamma$ is the eukaryotic counterpart of EF-Ts, acting as the guanine nucleotide exchange factor for EF-1$\alpha$. It facilitates the regeneration of EF-1$\alpha$-GTP by exchanging bound GDP for GTP.

Step-by-Step Mechanism of Elongation

Elongation proceeds cyclically through a series of precisely orchestrated steps for each amino acid added to the polypeptide chain:

Aminoacyl-tRNA Entry and Codon Recognition

The aminoacyl-tRNA, escorted by the ternary complex with EF-Tu (or EF-1$\alpha$) and GTP, enters the ribosomal A-site. Correct codon-anticodon base pairing between the mRNA and tRNA is essential at this stage.

GTP Hydrolysis and Proofreading

Hydrolysis of GTP bound to EF-Tu (or EF-1$\alpha$) occurs after codon recognition. This GTP hydrolysis is not only an energy source but also serves as a crucial proofreading step. It ensures that only after correct codon-anticodon interaction is confirmed, the elongation factor is released, and the aminoacyl-tRNA is properly accommodated in the A-site. This prevents premature entry of the tRNA acceptor stem into the catalytic site before proper codon verification.

Peptidyl Transferase Reaction

Once a correct aminoacyl-tRNA is in the A-site and the growing polypeptide chain is attached to tRNA in the P-site, the peptidyl transferase reaction is catalyzed.

Ribozyme Activity of rRNA

The peptidyl transferase activity is an inherent catalytic function of the ribosomal RNA (rRNA) within the large ribosomal subunit. Specifically, the 23S rRNA in prokaryotes and the 28S rRNA in eukaryotes are responsible for catalyzing peptide bond formation. This ribozyme activity underscores the critical role of RNA in ribosome function. The precise molecular mechanism of this RNA-catalyzed reaction is still under investigation, hampered by the difficulty in performing site-directed mutagenesis on rRNA without disrupting its structural integrity.

Peptide Bond Formation

In the peptidyl transferase reaction, the carboxyl group of the amino acid in the P-site (part of the nascent polypeptide chain) forms a peptide bond with the amino group of the amino acid in the A-site. This results in the transfer of the polypeptide chain from the tRNA in the P-site to the tRNA in the A-site, effectively adding one amino acid to the growing chain.

Antibiotic Inhibition of Elongation: Puromycin

Several antibiotics target the elongation phase, effectively inhibiting bacterial protein synthesis. Puromycin is a notable example.

Mechanism of Molecular Mimicry

Puromycin acts as a structural analog of aminoacyl-tRNA, specifically mimicking the aminoacyl-adenosine end of tRNA. This molecular mimicry allows puromycin to enter the ribosomal A-site.

Premature Termination of Polypeptide Synthesis

Once in the A-site, puromycin can participate in the peptidyl transferase reaction, accepting the growing polypeptide chain. However, unlike tRNA, puromycin lacks the necessary structure to engage with the P-site and participate in translocation. Consequently, the peptidyl-puromycin adduct is released prematurely from the ribosome, leading to the termination of translation and the production of truncated polypeptides. Puromycin is utilized in topical antibiotic ointments due to its antibacterial properties.

Translocation Phase

Overview of Translocation

Following peptide bond formation in the elongation cycle, the ribosome must advance along the mRNA to position the next codon into the A-site. This movement, termed translocation, is essential for continuous mRNA reading and subsequent polypeptide chain elongation. Translocation is an active process requiring energy derived from GTP hydrolysis.

Role of Elongation Factor G (EF-G)

Translocation is facilitated by Elongation Factor G (EF-G), known as EFG in prokaryotes and often referred to as EF-G in a general context.

GTP Hydrolysis and Energy Transduction

EF-G harnesses the energy from GTP hydrolysis to drive ribosome movement. This energy conversion is crucial for overcoming the kinetic barriers to translocation.

Molecular Mimicry and Mechanical Action

EF-G employs molecular mimicry, exhibiting a structural resemblance to tRNA. This structural similarity enables EF-G to interact with the ribosome in a manner analogous to tRNA. Functionally, EF-G acts as a "molecular crowbar," inserting itself into the ribosomal A-site to induce the mechanical movements necessary for translocation. This insertion promotes conformational changes within the ribosome structure.

Two-Step Mechanism of Ribosome Translocation

Ribosome translocation occurs through a two-step mechanism mediated by EF-G:

EF-G Insertion and Large Subunit Movement

EF-G initially inserts into the ribosomal A-site. This insertion triggers a significant conformational change primarily within the large ribosomal subunit. Driven by GTP hydrolysis, this conformational shift results in the forward movement of the large subunit by precisely one codon relative to the mRNA.

Small Subunit and mRNA Translocation

The movement of the large subunit is subsequently coupled to the movement of the small subunit. The forward shift of the large subunit effectively pulls the small subunit and the associated mRNA in the 3’ direction by one codon. This coordinated movement of both ribosomal subunits and the mRNA achieves the complete translocation of the ribosome to the next codon, ready for the next round of elongation.

Termination Phase

Overview of Termination

Termination is the concluding phase of protein synthesis, initiated when the ribosome encounters a stop codon on the mRNA. This phase culminates in the release of the fully synthesized polypeptide chain and the subsequent disassembly of the ribosome from the mRNA and tRNA molecules. Like elongation and translocation, termination is an energy-dependent process involving GTP hydrolysis.

Release Factors (RFs): Mediators of Termination

Polypeptide chain release and ribosome disassembly are mediated by proteins known as release factors (RFs). These factors recognize stop codons and orchestrate the events leading to termination.

Class 1 Release Factors: Stop Codon Recognition

Class 1 release factors are responsible for recognizing the stop codons (UAA, UAG, UGA) in the ribosomal A-site. This recognition event is the trigger for termination.

Prokaryotic RF1 and RF2: Codon-Specific Recognition

In prokaryotes, two Class 1 RFs exist, each with specificity for different stop codons:

RF1: Recognizes the stop codons UAA and UAG.
RF2: Recognizes the stop codons UAA and UGA.

Note that both RF1 and RF2 can recognize UAA, providing redundancy in stop codon recognition.

Eukaryotic eRF1: Universal Stop Codon Recognition

Eukaryotes utilize a single Class 1 release factor, eRF1 (eukaryotic Release Factor 1), which is capable of recognizing all three stop codons (UAA, UAG, and UGA). This contrasts with the codon-specific RFs in prokaryotes.

Class 2 Release Factors: Facilitating RF Release and Ribosome Recycling

Class 2 release factors are GTPases that play a catalytic role in termination. They do not directly recognize stop codons but are essential for the release of Class 1 RFs from the ribosome and for promoting ribosome disassembly.

RF3/eRF3: GTPase-Driven Release of Class 1 RFs

Prokaryotic RF3: Facilitates the release of RF1 or RF2 from the ribosome after peptide release has occurred. RF3 is a GTPase, and GTP hydrolysis is required for its function.
Eukaryotic eRF3: Analogous to prokaryotic RF3, eRF3 promotes the release of eRF1 from the ribosome in eukaryotes. eRF3 is also a GTPase, and its activity is coupled to the termination process.

Mechanism of Polypeptide Chain Termination

The termination process is a precise sequence of events:

Stop Codon Entry and RF Binding to the A-Site

When a stop codon (UAA, UAG, or UGA) translocates into the ribosomal A-site, it is recognized by a Class 1 release factor (RF1 or RF2 in prokaryotes; eRF1 in eukaryotes) instead of an aminoacyl-tRNA. The release factor binds in the A-site, mimicking the shape of a tRNA.

Water-Mediated Hydrolysis of Peptidyl-tRNA Bond

Class 1 release factors induce a change in the peptidyl transferase center of the ribosome. This alteration causes the catalytic center to become hydrolytic. Instead of catalyzing peptide bond formation with an incoming amino acid, the ribosome now catalyzes the hydrolysis of the ester bond linking the polypeptide chain to the tRNA in the P-site. In essence, a water molecule acts as the acceptor for the carboxyl group of the polypeptide chain, leading to its release.

Release of the Polypeptide Chain

The hydrolysis of the peptidyl-tRNA bond results in the release of the completed polypeptide chain from the ribosome. The ribosome now contains a deacylated tRNA in the P-site and a release factor in the A-site, with the mRNA still associated.

Ribosome Recycling and Disassembly

Following polypeptide release, the ribosomal subunits, mRNA, and tRNA must be dissociated to enable ribosome recycling for further translation initiation events. Ribosome Recycling Factor (RRF) plays a key role in this disassembly process.

RRF and EF-G Mediated Ribosome Disassembly

Ribosome recycling is facilitated by the combined action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G), along with GTP hydrolysis.

RRF Binding: RRF, structurally similar to tRNA, binds to the ribosomal A-site.
EF-G Interaction and GTP Hydrolysis: EF-G interacts with the ribosome and RRF. GTP hydrolysis by EF-G provides the energy for ribosome disassembly.
Ribosome Dissociation: EF-G, acting as a "molecular crowbar" once again, promotes conformational changes that lead to the dissociation of the 70S (or 80S) ribosome into its 30S and 50S (or 40S and 60S) subunits. The mRNA and tRNA are also released.

This ribosome recycling process ensures that ribosomal subunits are available for initiating new rounds of translation.

Energy Expenditure in Protein Synthesis

High Energy Demand of Protein Synthesis

Protein synthesis is a metabolically expensive process, reflecting its critical importance for cellular function and survival. The synthesis of each peptide bond is coupled to the hydrolysis of nucleoside triphosphates, primarily ATP and GTP. This section details the energy expenditure at each stage of translation.

ATP and GTP Consumption at Each Stage

The energy costs associated with protein synthesis can be broken down by stage:

Amino Acid Activation (tRNA Charging): Each amino acid must be activated and attached to its cognate tRNA. This aminoacylation process consumes 1 ATP per amino acid, which is hydrolyzed to AMP and PPi (pyrophosphate). The PPi is subsequently hydrolyzed to 2 Pi, further driving the reaction forward.
Initiation: The initiation phase, which involves ribosome assembly and mRNA binding, requires the hydrolysis of 2 GTP molecules in prokaryotes. Eukaryotic initiation, while more complex, has a comparable GTP consumption.
Elongation: Each cycle of elongation, which adds one amino acid to the polypeptide chain, requires the hydrolysis of 1 GTP molecule. This GTP is hydrolyzed during the delivery and proofreading of the aminoacyl-tRNA to the A-site by EF-Tu (or EF-1$\alpha$).
Translocation: The translocation step, moving the ribosome along the mRNA, consumes 1 GTP molecule per amino acid added. This GTP hydrolysis is associated with the function of EF-G (or EF-G).
Termination: The termination phase, leading to polypeptide release and ribosome dissociation, requires the hydrolysis of 1 GTP molecule. This GTP is utilized by the Class 2 release factor RF3 (or eRF3).
Ribosome Recycling: The recycling of ribosomal subunits for subsequent rounds of translation also involves GTP hydrolysis. The Ribosome Recycling Factor (RRF) and EF-G, in combination, utilize 1 GTP to dissociate the ribosome complex.

Overall Energy Cost Calculation

For a polypeptide chain of $N$ amino acids, the total energy cost can be approximated as follows:

ATP Consumption: $N$ ATP (for tRNA charging)
GTP Consumption: $2 \text{ (initiation)} + N \text{ (elongation)} + N \text{ (translocation)} + 1 \text{ (termination)} + 1 \text{ (recycling)} = 2N + 4$ GTP

Thus, the total high-energy phosphate bonds consumed are approximately $N \text{ ATP} + (2N + 4) \text{ GTP}$. For a protein of $N$ amino acids, the synthesis cost is roughly $4N + \text{constant}$ high-energy phosphate equivalents, where the constant accounts for the initiation, termination, and recycling costs, which are independent of protein length. For a typical human protein of approximately 400 amino acids, the energetic burden of synthesis is substantial, highlighting the significant allocation of cellular resources to protein production.

Antibiotics Targeting Translation

Broad Range of Translation Targets

While the elongation phase is a prominent target for antibiotics, exemplified by puromycin, antibacterial agents also effectively target other stages of translation, including initiation and termination. Furthermore, some antibiotics indirectly impact translation by inhibiting rRNA synthesis through targeting bacterial transcription machinery.

Challenges in Antibiotic Development: Selectivity

A major hurdle in developing new antibiotics is achieving selective toxicity against bacteria while minimizing harm to the eukaryotic host. The significant functional and structural conservation between prokaryotic and eukaryotic translation systems complicates the design of drugs that exclusively target bacterial processes without off-target effects in human cells.

Ada Yonath’s Contributions to Antibiotic Development

Professor Ada Yonath, a Nobel Laureate in Chemistry, has significantly advanced the field through her groundbreaking structural studies of the ribosome. Her detailed elucidation of ribosome structure and function has provided critical insights that have been instrumental in the rational design and development of novel antibiotics.

The Growing Threat of Antibiotic Resistance

The escalating prevalence of antibiotic resistance poses a critical global health challenge. Bacteria possess remarkable adaptability and evolve resistance mechanisms to circumvent the inhibitory effects of antibiotics. This necessitates ongoing research and development of new antibacterial strategies to combat resistant strains.

Examples of Antibiotic Classes and Mechanisms Targeting Translation

Several classes of antibiotics exert their antibacterial effects by targeting distinct steps in bacterial translation. The following table summarizes key examples and their mechanisms of action:

Examples of Antibiotics Targeting Translation and Related Processes
Antibiotic Class	Example	Mechanism of Action
Tetracyclines	Tetracycline	Block aminoacyl-tRNA binding to the ribosomal A-site, inhibiting elongation.
Aminoglycosides	Streptomycin	Interfere with the transition from initiation to elongation; induce mRNA misreading at lower concentrations; block initiation at higher concentrations.
Macrolides	Erythromycin	Bind to the ribosome exit tunnel, sterically hindering the exit of the nascent polypeptide and blocking elongation.
Antibiotics Affecting Related Processes
Rifamycins	Rifampicin	Inhibit bacterial RNA synthesis by targeting bacterial RNA polymerase, reducing mRNA availability for translation. (Transcription inhibitor)
Actinomycins	Actinomycin D	Inhibit RNA synthesis, but with broader and less specific effects, impacting both prokaryotic and eukaryotic transcription. (Transcription inhibitor, broad toxicity)
Puromycin	Puromycin	Cause premature termination of translation by acting as an aminoacyl-tRNA analog. (Mimics tRNA, premature termination)

Side Effects and Mitochondrial Toxicity of Antibiotics

Certain antibiotics can elicit side effects due to their off-target toxicity towards mitochondria. Mitochondrial ribosomes exhibit greater structural similarity to bacterial ribosomes than to cytoplasmic ribosomes in eukaryotes. Consequently, antibiotics designed to target bacterial ribosomes can inadvertently interfere with mitochondrial protein synthesis. This interference can lead to side effects such as fatigue and muscle weakness, particularly affecting tissues with high mitochondrial density like muscle. Furthermore, disruption of the gut microbiota is a common side effect associated with many antibiotics, as they can also target beneficial bacteria in the gut.

Mechanisms of Bacterial Resistance to Antibiotics

Bacteria have evolved diverse mechanisms to develop resistance against antibiotics, compromising their effectiveness. Key resistance mechanisms include:

Mutations in Target Genes

Mutations in bacterial genes encoding ribosomal RNA (rRNA), ribosomal proteins, or elongation factors can alter the antibiotic binding site on the ribosome. These mutations reduce the affinity of the antibiotic for its target, thereby diminishing or abolishing its inhibitory effect.

Efflux Pumps and Reduced Drug Uptake

Bacteria can express efflux pumps, membrane-associated proteins that actively transport antibiotics out of the bacterial cell. This reduces the intracellular concentration of the antibiotic, rendering it ineffective. Additionally, alterations in porin channels in the bacterial outer membrane can restrict antibiotic entry into the cell, further contributing to resistance. These efflux and reduced uptake mechanisms are also observed in multidrug resistance in tumor cells, highlighting convergent strategies for drug evasion in different biological contexts.

Introduction to DNA Repair and Genomic Stability

Transition to DNA Repair Mechanisms

Having concluded our discussion on the intricate process of translation, we now turn our attention to DNA repair and the maintenance of genomic stability. This is a critical domain for understanding cellular physiology, disease pathogenesis, and notably, cancer biology.

The Paramount Importance of Genomic Stability

Genomic stability is essential for the accurate inheritance of genetic information across generations and for the proper functioning of individual cells within an organism. DNA uniquely serves as the repository of heritable information and, unlike other biological macromolecules such as RNA, proteins, lipids, and polysaccharides that undergo metabolic turnover, DNA is actively maintained and repaired to preserve its integrity rather than being replaced.

DNA Repair Defects and Disease: Focus on Cancer

Dysfunction in DNA repair mechanisms is implicated in a spectrum of diseases, with cancer being the most prominent. A thorough understanding of DNA repair processes is therefore indispensable for elucidating cancer development, progression, and therapeutic interventions.

Ubiquitous DNA Damage and Cellular Repair Systems

Cells are continuously subjected to DNA-damaging agents originating from both internal metabolic activities (endogenous sources) and external environmental exposures (exogenous sources). To counteract the constant threat of DNA damage, cells have evolved a complex and sophisticated network of DNA repair pathways. These pathways are crucial for preserving the integrity of the genome and preventing the accumulation of mutations that can lead to cellular dysfunction and disease.

Fundamental Principles of DNA Repair

DNA Integrity and Hereditary Information

DNA serves as the fundamental repository of hereditary information, making its structural and informational integrity paramount for life. The substantial energetic investment cells make in DNA repair mechanisms underscores the critical importance of maintaining the genome.

Energetic Investment in DNA Repair Systems

The complexity and scope of DNA repair are reflected in the large number of proteins—hundreds—dedicated to these processes. This extensive molecular machinery represents a significant energetic commitment by the cell, comparable to the energy expenditure for essential processes like transcription and translation. This high energy cost highlights the indispensable role of DNA repair in ensuring cell survival and genomic fidelity.

Distinction Between DNA Lesions and Mutations

Definition 1 (DNA Lesion). A DNA lesion is a physical or chemical alteration to the DNA molecule. This alteration can be a base modification, strand break, or other forms of damage.

Definition 2 (Mutation). A DNA lesion becomes a mutation only when it is not repaired before DNA replication and results in a permanent change in the DNA sequence that is heritable in subsequent generations. Most DNA lesions are repaired before replication can fix them into mutations.

Understanding this distinction is essential, as not all DNA damage events lead to permanent genetic changes.

General Principles of DNA Repair Pathways

Several overarching principles govern the operation of DNA repair pathways, ensuring their effectiveness and fidelity.

Specificity of Lesion Recognition

DNA repair pathways exhibit remarkable specificity. Different pathways have evolved to recognize and repair distinct types of DNA lesions. This specialization reflects the diverse nature of DNA damage caused by various endogenous and exogenous agents.

Pathway Redundancy and Interconnection

Redundancy is a key feature of DNA repair systems. Multiple pathways may exist to repair the same type of DNA damage, and enzymes can participate in more than one repair pathway. Furthermore, repair intermediates generated by one pathway can serve as substrates for other pathways. This interconnected and redundant network ensures robust and efficient DNA repair, maximizing the chances of correcting DNA damage. For instance, pyrimidine dimers can be processed by both error-prone translesion synthesis and the high-fidelity Nucleotide Excision Repair (NER) pathway.

Utilization of Complementary Strand Information

The majority of DNA repair pathways leverage the information encoded in the undamaged complementary DNA strand to guide accurate repair. The double-helix structure of DNA, with its inherent base complementarity, is thus fundamental to high-fidelity DNA repair. The undamaged strand serves as a template to restore the correct sequence in the damaged strand.

Frequency and Efficiency of DNA Repair in Human Cells

Human cells are constantly challenged by a high frequency of DNA lesions. It is estimated that each cell in the human body sustains between $10^4$ and $10^6$ DNA lesions per day due to normal metabolic processes and environmental exposures. This translates to an astonishing $10^{18}$ to $10^{20}$ DNA repair events occurring daily throughout the human body. Despite this substantial burden of DNA damage, cellular repair mechanisms are remarkably efficient. Less than one in every 1000 DNA lesions, on average, escapes repair and becomes a permanent mutation. This high efficiency of DNA repair is crucial for maintaining genomic stability and preventing disease.

DNA Damage Tolerance vs. Repair Mechanisms

Cells employ a combination of DNA repair and damage tolerance mechanisms to manage DNA lesions.

Error-Prone Translesion Synthesis (TLS) Polymerases

A subset of DNA polymerases, known as translesion synthesis (TLS) polymerases (e.g., polymerase $\eta$, $\iota$, $\zeta$, $\kappa$, $\lambda$, $\mu$), are specialized for replicating past DNA lesions that would otherwise stall replicative polymerases. These TLS polymerases typically lack proofreading activity and are error-prone. While they allow DNA replication to proceed in the presence of damage, they often introduce mutations at or near the lesion site. Thus, TLS is considered a DNA damage tolerance mechanism rather than a true repair pathway, as it prioritizes replication completion over maintaining the original DNA sequence.

Error-Prone Non-Homologous End Joining (NHEJ)

Non-Homologous End Joining (NHEJ) is a major pathway for repairing DNA double-strand breaks (DSBs). While NHEJ can efficiently rejoin broken DNA ends, it is considered error-prone because it often involves small insertions or deletions at the repair site. NHEJ directly ligates the DNA ends without using a homologous template, which can lead to loss of genetic information and introduce mutations. Despite its error-prone nature, NHEJ is essential for repairing DSBs, which are particularly hazardous lesions that can lead to chromosomal instability and cell death if left unrepaired.

DNA Repair and Cancer

Causal Link Between DNA Damage and Cancer

A significant majority of cancers, approximately 80%, are etiologically linked to DNA-damaging agents. This strong association underscores the critical role of DNA damage in the initiation and progression of cancer.

Age-Dependent Cancer Risk and Cumulative DNA Damage

The incidence of cancer exhibits an exponential increase with age, rather than a linear progression. This exponential relationship reflects the cumulative impact of DNA damage over an individual’s lifespan and the progressive accumulation of mutations through successive cell divisions. Increased age correlates with prolonged exposure to mutagens and a greater number of cell cycles, thereby elevating the probability of mutation fixation and subsequent tumorigenesis.

Hereditary Cancer Predisposition Syndromes: Defects in DNA Repair Genes

Numerous hereditary cancer syndromes are directly attributable to germline mutations in genes encoding components of DNA repair pathways. These inherited mutations compromise DNA repair capacity, predisposing individuals to a heightened risk of developing specific types of cancer.

Exemplary Cancer Predisposition Syndromes and Defective Repair Pathways

Several well-characterized cancer predisposition syndromes serve as compelling examples of the link between DNA repair defects and cancer susceptibility.

Xeroderma Pigmentosum (XP): Nucleotide Excision Repair (NER) Deficiency

Xeroderma Pigmentosum (XP) arises from mutations in genes involved in the Nucleotide Excision Repair (NER) pathway. NER is crucial for repairing bulky DNA lesions, including pyrimidine dimers induced by ultraviolet (UV) radiation. Individuals with XP exhibit extreme sensitivity to sunlight and have a markedly elevated risk of skin cancers due to their inability to efficiently repair UV-induced DNA damage.

BRCA1/BRCA2-Associated Cancer: Homologous Recombination (HR) Deficiency

Mutations in the BRCA1 and BRCA2 genes, key players in the Homologous Recombination (HR) DNA repair pathway, are strongly associated with an increased susceptibility to breast and ovarian cancers, particularly in women. BRCA1 and BRCA2 are essential for the accurate repair of DNA double-strand breaks. The widespread public awareness of BRCA gene mutations has been significantly amplified by personal accounts and preventative health decisions. BRCA1 and BRCA2 mutations are clinically relevant as both prognostic and predictive biomarkers in cancer management. Furthermore, the germline nature of these mutations has implications for genetic counseling and family planning due to the potential for vertical transmission to offspring.

Hereditary Non-Polyposis Colorectal Cancer (HNPCC) / Lynch Syndrome: Mismatch Repair (MMR) Deficiency

Hereditary Non-Polyposis Colorectal Cancer (HNPCC), also known as Lynch Syndrome, is caused by inherited mutations in genes of the Mismatch Repair (MMR) pathway. MMR is critical for correcting base-base mismatches and insertion/deletion loops that arise during DNA replication. Individuals with HNPCC have a pronounced predisposition to colorectal cancer and other gastrointestinal malignancies. The high proliferative rate and rapid turnover of intestinal epithelial cells render them particularly vulnerable to replication errors, highlighting their dependence on functional MMR.

Driver and Passenger Mutations in Cancer Development

Mutations implicated in cancer development are broadly categorized as either driver or passenger mutations, based on their functional roles in tumorigenesis.

Definition 3 (Driver Mutations). Driver mutations, also termed founder mutations, are mutations that directly initiate and drive the process of tumorigenesis. These mutations typically occur early in cancer development and affect genes that are critically involved in regulating cell growth, survival, and genomic stability. Genes encoding DNA repair proteins are frequently targets of founder mutations, as their disruption can lead to genomic instability, a hallmark of cancer.

Definition 4 (Passenger Mutations). Passenger mutations, in contrast, are mutations that accumulate during tumor progression but do not directly initiate tumorigenesis. However, a subset of passenger mutations, termed progressor mutations or essential passenger mutations, confer selective advantages to tumor cells, contributing to tumor evolution, metastasis, and drug resistance. These mutations often arise as a consequence of the genomic instability initiated by founder mutations, enabling tumor cells to adapt and evolve within the selective pressures of the tumor microenvironment and therapeutic interventions.

DNA Repair Genes as Frequent Targets of Founder Mutations

Genes responsible for maintaining genomic stability, notably DNA repair genes, are frequently targeted by founder mutations in cancer. Examples of such genes include p53 (a central tumor suppressor involved in DNA damage response and repair), cell cycle regulatory genes (such as CDKN2A), and proto-oncogenes (like K-ras). Mutations in these genes disrupt fundamental cellular control mechanisms, initiating acascade of events that can lead to uncontrolled cell proliferation and tumor development.

Cellular Origin of Tumors: The Cancer Stem Cell Hypothesis

The cancer stem cell hypothesis posits that tumors originate from a subpopulation of cells within the tumor termed cancer stem cells (CSCs). These cells possess stem-cell-like properties, including self-renewal capacity and the ability to differentiate into various cell types within the tumor. CSCs are proposed to be responsible for tumor initiation, sustained tumor growth, metastasis, and recurrence after therapy.

Genomic Instability as an Enabling Hallmark of Cancer

The "hallmarks of cancer" represent a conceptual framework describing the essential biological capabilities acquired by cancer cells during tumorigenesis. Genomic instability is now recognized as an enabling hallmark that underpins the acquisition of many other hallmarks.

Genomic Instability: Fueling Tumor Evolution and Heterogeneity

Genomic instability, characterized by an increased rate of mutations and chromosomal aberrations, is not merely a consequence of tumorigenesis but actively facilitates it. In the early stages of tumor development, genomic instability promotes the accumulation of driver mutations that initiate and accelerate tumor evolution. However, while initially promoting genetic diversity and adaptation, excessive genomic instability can be detrimental, leading to cell death or growth arrest. Therefore, cancer cells must strike a balance, maintaining a level of genomic instability that is sufficient to drive evolution and adaptation but not so extreme as to compromise cell viability.

Interplay of Hallmarks and the Role of Genomic Instability

The various hallmarks of cancer are interconnected and operate synergistically to drive tumor development and progression. Genomic instability acts as a critical enabler, facilitating the acquisition and manifestation of other hallmarks, including sustained proliferative signaling, evasion of growth suppressors, resistance to cell death (apoptosis), induction of angiogenesis, and activation of invasion and metastasis. Chronic inflammation, often present in the tumor microenvironment, further contributes to genomic instability by producing reactive oxygen species and inflammatory mediators that can damage DNA, creating a vicious cycle that promotes tumorigenesis.

Intracellular Signaling Circuits and Key Cancer Genes

Intracellular signaling circuits involving proto-oncogenes (e.g., Ras, Myc), tumor suppressor genes (e.g., p53), and cell cycle regulators (e.g., CDKN2A) are frequently dysregulated in cancer cells, contributing to the hallmark capabilities of cancer.

Key Regulatory Genes in Cancer: p53, Cell Cycle Regulators, Proto-oncogenes

p53: A central tumor suppressor gene, p53 is among the most frequently mutated genes in human cancers. Mutant p53 proteins often exhibit oncogenic or oncomorphic gain-of-function activities, further promoting tumorigenesis.
Cell Cycle Regulators (e.g., CDKN2A): Genes that control cell cycle progression are frequently mutated or dysregulated in cancer, leading to uncontrolled proliferation. CDKN2A, encoding p16INK4a and p14ARF, is a key tumor suppressor locus often inactivated in various cancers.
Proto-oncogenes (e.g., Ras, Myc): Proto-oncogenes, when mutated or overexpressed, can become oncogenes that drive cell proliferation and survival. Ras family GTPases and Myc transcription factors are prominent examples of proto-oncogenes frequently activated in cancer.

Therapeutic Implications of Targeting Founder Mutations

From a therapeutic perspective, targeting founder mutations represents the most effective strategy for achieving durable cancer eradication.However, tumors are often diagnosed at later stages, when progressor mutations and intratumoral heterogeneity are already established. This clonal evolution and heterogeneity pose significant challenges to therapy, as treatments targeting progressor mutations may encounter resistance due to the selective outgrowth of pre-existing resistant subclones within the tumor. A comprehensive understanding of the evolutionary dynamics of tumor development, the temporal order of mutation acquisition, and the distinct roles of founder and progressor mutations is crucial for developing more effective and personalized cancer therapies that can overcome resistance and improve patient outcomes.

Conclusion

In summary, this lecture has detailed the elongation, translocation, and termination phases of translation, emphasizing the energy expenditure inherent in protein synthesis and the mechanisms of action of antibiotics that target translation. We then transitioned to an introduction to DNA repair and genomic stability, underscoring the vital role of DNA repair in preventing mutations and cancer. We discussed the fundamental principles of DNA repair, the diverse types of DNA damage, and the established link between deficiencies in DNA repair pathways and cancer predisposition syndromes. We introduced the concepts of driver and passenger mutations in cancer development and the hallmarks of cancer, setting the stage for a more in-depth exploration of specific DNA repair pathways and their functions in maintaining genomic integrity and preventing tumorigenesis in subsequent lectures. The next lecture will delve into the specific types of DNA damage and the detailed mechanisms of various DNA repair pathways.

--- title: "Lecture Notes on Translation and DNA Repair" author: "Your Name" date: "2025-02-05" format: html: toc: true # Table of Contents toc-depth: 2 code-tools: true theme: cosmo # Or "journal" for Distill-like minimalism --- # Introduction In the preceding lecture, we examined the initiation phase of translation, emphasizing the distinctions between prokaryotic and eukaryotic systems. This lecture will conclude our exploration of translation by detailing the elongation and termination phases. We will note the conserved nature of the elongation process across prokaryotes and eukaryotes. Subsequently, we will introduce the topic of DNA repair and genomic stability, laying the groundwork for future discussions in this area. # Elongation Phase ## Overview of Elongation The elongation phase of translation is characterized by the sequential addition of amino acids to a growing polypeptide chain. This process is highly conserved between prokaryotic and eukaryotic organisms, indicating its fundamental importance in cellular life. ## Key Components in Elongation The elongation phase necessitates the coordinated function of several components: - **Ribosomes:** Serve as the central machinery, providing the structural framework and catalytic activity for peptide bond formation. - **Aminoacyl-tRNAs:** Act as carriers of amino acids, delivering them to the ribosome according to the mRNA sequence. - **Elongation Factors (EFs):** Accessory proteins that facilitate and regulate the various steps of elongation, enhancing both the rate and accuracy of protein synthesis. ## Elongation Factors: Types and Function Elongation factors are crucial for the efficient and accurate progression of the elongation phase. They ensure correct aminoacyl-tRNA delivery, utilize GTP hydrolysis for energy and proofreading, and facilitate ribosome translocation. ### Prokaryotic Elongation Factors Prokaryotes utilize two primary elongation factors: EF-Tu and EF-Ts. #### EF-Tu EF-Tu (Elongation Factor Thermo unstable) is responsible for the delivery of aminoacyl-tRNA to the ribosomal A-site. This delivery is facilitated by the formation of a ternary complex consisting of EF-Tu, GTP, and aminoacyl-tRNA. GTP hydrolysis by EF-Tu is essential for its function and for proofreading during codon recognition. #### EF-Ts EF-Ts (Elongation Factor Thermo stable) functions as a guanine nucleotide exchange factor (GEF). It recycles EF-Tu by catalyzing the exchange of GDP for GTP, thus regenerating the active EF-Tu-GTP form required for subsequent elongation cycles. ### Eukaryotic Elongation Factors Eukaryotes employ analogous elongation factors to their prokaryotic counterparts. #### EF-1$\alpha$ EF-1$\alpha$ is the eukaryotic functional ortholog of EF-Tu. It performs the same role of delivering aminoacyl-tRNA to the ribosome A-site as a ternary complex with GTP and aminoacyl-tRNA, coupled with GTP hydrolysis. #### EF-1$\beta\gamma$ EF-1$\beta\gamma$ is the eukaryotic counterpart of EF-Ts, acting as the guanine nucleotide exchange factor for EF-1$\alpha$. It facilitates the regeneration of EF-1$\alpha$-GTP by exchanging bound GDP for GTP. ## Step-by-Step Mechanism of Elongation Elongation proceeds cyclically through a series of precisely orchestrated steps for each amino acid added to the polypeptide chain: ### Aminoacyl-tRNA Entry and Codon Recognition The aminoacyl-tRNA, escorted by the ternary complex with EF-Tu (or EF-1$\alpha$) and GTP, enters the ribosomal A-site. Correct codon-anticodon base pairing between the mRNA and tRNA is essential at this stage. ### GTP Hydrolysis and Proofreading Hydrolysis of GTP bound to EF-Tu (or EF-1$\alpha$) occurs after codon recognition. This GTP hydrolysis is not only an energy source but also serves as a crucial proofreading step. It ensures that only after correct codon-anticodon interaction is confirmed, the elongation factor is released, and the aminoacyl-tRNA is properly accommodated in the A-site. This prevents premature entry of the tRNA acceptor stem into the catalytic site before proper codon verification. ### Peptidyl Transferase Reaction Once a correct aminoacyl-tRNA is in the A-site and the growing polypeptide chain is attached to tRNA in the P-site, the peptidyl transferase reaction is catalyzed. #### Ribozyme Activity of rRNA The peptidyl transferase activity is an inherent catalytic function of the ribosomal RNA (rRNA) within the large ribosomal subunit. Specifically, the 23S rRNA in prokaryotes and the 28S rRNA in eukaryotes are responsible for catalyzing peptide bond formation. This ribozyme activity underscores the critical role of RNA in ribosome function. The precise molecular mechanism of this RNA-catalyzed reaction is still under investigation, hampered by the difficulty in performing site-directed mutagenesis on rRNA without disrupting its structural integrity. #### Peptide Bond Formation In the peptidyl transferase reaction, the carboxyl group of the amino acid in the P-site (part of the nascent polypeptide chain) forms a peptide bond with the amino group of the amino acid in the A-site. This results in the transfer of the polypeptide chain from the tRNA in the P-site to the tRNA in the A-site, effectively adding one amino acid to the growing chain. ## Antibiotic Inhibition of Elongation: Puromycin Several antibiotics target the elongation phase, effectively inhibiting bacterial protein synthesis. Puromycin is a notable example. ### Mechanism of Molecular Mimicry Puromycin acts as a structural analog of aminoacyl-tRNA, specifically mimicking the aminoacyl-adenosine end of tRNA. This molecular mimicry allows puromycin to enter the ribosomal A-site. ### Premature Termination of Polypeptide Synthesis Once in the A-site, puromycin can participate in the peptidyl transferase reaction, accepting the growing polypeptide chain. However, unlike tRNA, puromycin lacks the necessary structure to engage with the P-site and participate in translocation. Consequently, the peptidyl-puromycin adduct is released prematurely from the ribosome, leading to the termination of translation and the production of truncated polypeptides. Puromycin is utilized in topical antibiotic ointments due to its antibacterial properties. ![Mechanism of Puromycin Action. Puromycin, resembling aminoacyl-tRNA, causes premature termination of polypeptide synthesis by entering the A-site and releasing a peptidyl-puromycin adduct.](puromycin_mechanism.png){#fig:puromycin width="50%"} # Translocation Phase ## Overview of Translocation Following peptide bond formation in the elongation cycle, the ribosome must advance along the mRNA to position the next codon into the A-site. This movement, termed translocation, is essential for continuous mRNA reading and subsequent polypeptide chain elongation. Translocation is an active process requiring energy derived from GTP hydrolysis. ## Role of Elongation Factor G (EF-G) Translocation is facilitated by Elongation Factor G (EF-G), known as EFG in prokaryotes and often referred to as EF-G in a general context. ### GTP Hydrolysis and Energy Transduction EF-G harnesses the energy from GTP hydrolysis to drive ribosome movement. This energy conversion is crucial for overcoming the kinetic barriers to translocation. ### Molecular Mimicry and Mechanical Action EF-G employs molecular mimicry, exhibiting a structural resemblance to tRNA. This structural similarity enables EF-G to interact with the ribosome in a manner analogous to tRNA. Functionally, EF-G acts as a \"molecular crowbar,\" inserting itself into the ribosomal A-site to induce the mechanical movements necessary for translocation. This insertion promotes conformational changes within the ribosome structure. ## Two-Step Mechanism of Ribosome Translocation Ribosome translocation occurs through a two-step mechanism mediated by EF-G: ### EF-G Insertion and Large Subunit Movement EF-G initially inserts into the ribosomal A-site. This insertion triggers a significant conformational change primarily within the large ribosomal subunit. Driven by GTP hydrolysis, this conformational shift results in the forward movement of the large subunit by precisely one codon relative to the mRNA. ### Small Subunit and mRNA Translocation The movement of the large subunit is subsequently coupled to the movement of the small subunit. The forward shift of the large subunit effectively pulls the small subunit and the associated mRNA in the 3' direction by one codon. This coordinated movement of both ribosomal subunits and the mRNA achieves the complete translocation of the ribosome to the next codon, ready for the next round of elongation. # Termination Phase ## Overview of Termination Termination is the concluding phase of protein synthesis, initiated when the ribosome encounters a stop codon on the mRNA. This phase culminates in the release of the fully synthesized polypeptide chain and the subsequent disassembly of the ribosome from the mRNA and tRNA molecules. Like elongation and translocation, termination is an energy-dependent process involving GTP hydrolysis. ## Release Factors (RFs): Mediators of Termination Polypeptide chain release and ribosome disassembly are mediated by proteins known as release factors (RFs). These factors recognize stop codons and orchestrate the events leading to termination. ### Class 1 Release Factors: Stop Codon Recognition Class 1 release factors are responsible for recognizing the stop codons (UAA, UAG, UGA) in the ribosomal A-site. This recognition event is the trigger for termination. #### Prokaryotic RF1 and RF2: Codon-Specific Recognition In prokaryotes, two Class 1 RFs exist, each with specificity for different stop codons: - **RF1:** Recognizes the stop codons UAA and UAG. - **RF2:** Recognizes the stop codons UAA and UGA. Note that both RF1 and RF2 can recognize UAA, providing redundancy in stop codon recognition. #### Eukaryotic eRF1: Universal Stop Codon Recognition Eukaryotes utilize a single Class 1 release factor, eRF1 (eukaryotic Release Factor 1), which is capable of recognizing all three stop codons (UAA, UAG, and UGA). This contrasts with the codon-specific RFs in prokaryotes. ### Class 2 Release Factors: Facilitating RF Release and Ribosome Recycling Class 2 release factors are GTPases that play a catalytic role in termination. They do not directly recognize stop codons but are essential for the release of Class 1 RFs from the ribosome and for promoting ribosome disassembly. #### RF3/eRF3: GTPase-Driven Release of Class 1 RFs - **Prokaryotic RF3:** Facilitates the release of RF1 or RF2 from the ribosome after peptide release has occurred. RF3 is a GTPase, and GTP hydrolysis is required for its function. - **Eukaryotic eRF3:** Analogous to prokaryotic RF3, eRF3 promotes the release of eRF1 from the ribosome in eukaryotes. eRF3 is also a GTPase, and its activity is coupled to the termination process. ## Mechanism of Polypeptide Chain Termination The termination process is a precise sequence of events: ### Stop Codon Entry and RF Binding to the A-Site When a stop codon (UAA, UAG, or UGA) translocates into the ribosomal A-site, it is recognized by a Class 1 release factor (RF1 or RF2 in prokaryotes; eRF1 in eukaryotes) instead of an aminoacyl-tRNA. The release factor binds in the A-site, mimicking the shape of a tRNA. ### Water-Mediated Hydrolysis of Peptidyl-tRNA Bond Class 1 release factors induce a change in the peptidyl transferase center of the ribosome. This alteration causes the catalytic center to become hydrolytic. Instead of catalyzing peptide bond formation with an incoming amino acid, the ribosome now catalyzes the hydrolysis of the ester bond linking the polypeptide chain to the tRNA in the P-site. In essence, a water molecule acts as the acceptor for the carboxyl group of the polypeptide chain, leading to its release. ### Release of the Polypeptide Chain The hydrolysis of the peptidyl-tRNA bond results in the release of the completed polypeptide chain from the ribosome. The ribosome now contains a deacylated tRNA in the P-site and a release factor in the A-site, with the mRNA still associated. ## Ribosome Recycling and Disassembly Following polypeptide release, the ribosomal subunits, mRNA, and tRNA must be dissociated to enable ribosome recycling for further translation initiation events. Ribosome Recycling Factor (RRF) plays a key role in this disassembly process. ### RRF and EF-G Mediated Ribosome Disassembly Ribosome recycling is facilitated by the combined action of Ribosome Recycling Factor (RRF) and Elongation Factor G (EF-G), along with GTP hydrolysis. - **RRF Binding:** RRF, structurally similar to tRNA, binds to the ribosomal A-site. - **EF-G Interaction and GTP Hydrolysis:** EF-G interacts with the ribosome and RRF. GTP hydrolysis by EF-G provides the energy for ribosome disassembly. - **Ribosome Dissociation:** EF-G, acting as a \"molecular crowbar\" once again, promotes conformational changes that lead to the dissociation of the 70S (or 80S) ribosome into its 30S and 50S (or 40S and 60S) subunits. The mRNA and tRNA are also released. This ribosome recycling process ensures that ribosomal subunits are available for initiating new rounds of translation. # Energy Expenditure in Protein Synthesis ## High Energy Demand of Protein Synthesis Protein synthesis is a metabolically expensive process, reflecting its critical importance for cellular function and survival. The synthesis of each peptide bond is coupled to the hydrolysis of nucleoside triphosphates, primarily ATP and GTP. This section details the energy expenditure at each stage of translation. ## ATP and GTP Consumption at Each Stage The energy costs associated with protein synthesis can be broken down by stage: - **Amino Acid Activation (tRNA Charging):** Each amino acid must be activated and attached to its cognate tRNA. This aminoacylation process consumes **1 ATP** per amino acid, which is hydrolyzed to AMP and PPi (pyrophosphate). The PPi is subsequently hydrolyzed to 2 Pi, further driving the reaction forward. - **Initiation:** The initiation phase, which involves ribosome assembly and mRNA binding, requires the hydrolysis of **2 GTP** molecules in prokaryotes. Eukaryotic initiation, while more complex, has a comparable GTP consumption. - **Elongation:** Each cycle of elongation, which adds one amino acid to the polypeptide chain, requires the hydrolysis of **1 GTP** molecule. This GTP is hydrolyzed during the delivery and proofreading of the aminoacyl-tRNA to the A-site by EF-Tu (or EF-1$\alpha$). - **Translocation:** The translocation step, moving the ribosome along the mRNA, consumes **1 GTP** molecule per amino acid added. This GTP hydrolysis is associated with the function of EF-G (or EF-G). - **Termination:** The termination phase, leading to polypeptide release and ribosome dissociation, requires the hydrolysis of **1 GTP** molecule. This GTP is utilized by the Class 2 release factor RF3 (or eRF3). - **Ribosome Recycling:** The recycling of ribosomal subunits for subsequent rounds of translation also involves GTP hydrolysis. The Ribosome Recycling Factor (RRF) and EF-G, in combination, utilize **1 GTP** to dissociate the ribosome complex. ## Overall Energy Cost Calculation For a polypeptide chain of $N$ amino acids, the total energy cost can be approximated as follows: - ATP Consumption: $N$ ATP (for tRNA charging) - GTP Consumption: $2 \text{ (initiation)} + N \text{ (elongation)} + N \text{ (translocation)} + 1 \text{ (termination)} + 1 \text{ (recycling)} = 2N + 4$ GTP Thus, the total high-energy phosphate bonds consumed are approximately $N \text{ ATP} + (2N + 4) \text{ GTP}$. For a protein of $N$ amino acids, the synthesis cost is roughly **$4N + \text{constant}$** high-energy phosphate equivalents, where the constant accounts for the initiation, termination, and recycling costs, which are independent of protein length. For a typical human protein of approximately 400 amino acids, the energetic burden of synthesis is substantial, highlighting the significant allocation of cellular resources to protein production. # Antibiotics Targeting Translation ## Broad Range of Translation Targets While the elongation phase is a prominent target for antibiotics, exemplified by puromycin, antibacterial agents also effectively target other stages of translation, including initiation and termination. Furthermore, some antibiotics indirectly impact translation by inhibiting rRNA synthesis through targeting bacterial transcription machinery. ## Challenges in Antibiotic Development: Selectivity A major hurdle in developing new antibiotics is achieving selective toxicity against bacteria while minimizing harm to the eukaryotic host. The significant functional and structural conservation between prokaryotic and eukaryotic translation systems complicates the design of drugs that exclusively target bacterial processes without off-target effects in human cells. ## Ada Yonath's Contributions to Antibiotic Development Professor Ada Yonath, a Nobel Laureate in Chemistry, has significantly advanced the field through her groundbreaking structural studies of the ribosome. Her detailed elucidation of ribosome structure and function has provided critical insights that have been instrumental in the rational design and development of novel antibiotics. ## The Growing Threat of Antibiotic Resistance The escalating prevalence of antibiotic resistance poses a critical global health challenge. Bacteria possess remarkable adaptability and evolve resistance mechanisms to circumvent the inhibitory effects of antibiotics. This necessitates ongoing research and development of new antibacterial strategies to combat resistant strains. ## Examples of Antibiotic Classes and Mechanisms Targeting Translation Several classes of antibiotics exert their antibacterial effects by targeting distinct steps in bacterial translation. The following table summarizes key examples and their mechanisms of action: ::: tab:antibiotics **Antibiotic Class** **Example** **Mechanism of Action** --------------------------------------------- --------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Tetracyclines** Tetracycline Block aminoacyl-tRNA binding to the ribosomal A-site, inhibiting elongation. **Aminoglycosides** Streptomycin Interfere with the transition from initiation to elongation; induce mRNA misreading at lower concentrations; block initiation at higher concentrations. **Macrolides** Erythromycin Bind to the ribosome exit tunnel, sterically hindering the exit of the nascent polypeptide and blocking elongation. **Antibiotics Affecting Related Processes** **Rifamycins** Rifampicin Inhibit bacterial RNA synthesis by targeting bacterial RNA polymerase, reducing mRNA availability for translation. (Transcription inhibitor) **Actinomycins** Actinomycin D Inhibit RNA synthesis, but with broader and less specific effects, impacting both prokaryotic and eukaryotic transcription. (Transcription inhibitor, broad toxicity) **Puromycin** Puromycin Cause premature termination of translation by acting as an aminoacyl-tRNA analog. (Mimics tRNA, premature termination) : Examples of Antibiotics Targeting Translation and Related Processes ::: ## Side Effects and Mitochondrial Toxicity of Antibiotics Certain antibiotics can elicit side effects due to their off-target toxicity towards mitochondria. Mitochondrial ribosomes exhibit greater structural similarity to bacterial ribosomes than to cytoplasmic ribosomes in eukaryotes. Consequently, antibiotics designed to target bacterial ribosomes can inadvertently interfere with mitochondrial protein synthesis. This interference can lead to side effects such as fatigue and muscle weakness, particularly affecting tissues with high mitochondrial density like muscle. Furthermore, disruption of the gut microbiota is a common side effect associated with many antibiotics, as they can also target beneficial bacteria in the gut. ## Mechanisms of Bacterial Resistance to Antibiotics Bacteria have evolved diverse mechanisms to develop resistance against antibiotics, compromising their effectiveness. Key resistance mechanisms include: ### Mutations in Target Genes Mutations in bacterial genes encoding ribosomal RNA (rRNA), ribosomal proteins, or elongation factors can alter the antibiotic binding site on the ribosome. These mutations reduce the affinity of the antibiotic for its target, thereby diminishing or abolishing its inhibitory effect. ### Efflux Pumps and Reduced Drug Uptake Bacteria can express efflux pumps, membrane-associated proteins that actively transport antibiotics out of the bacterial cell. This reduces the intracellular concentration of the antibiotic, rendering it ineffective. Additionally, alterations in porin channels in the bacterial outer membrane can restrict antibiotic entry into the cell, further contributing to resistance. These efflux and reduced uptake mechanisms are also observed in multidrug resistance in tumor cells, highlighting convergent strategies for drug evasion in different biological contexts. # Introduction to DNA Repair and Genomic Stability ## Transition to DNA Repair Mechanisms Having concluded our discussion on the intricate process of translation, we now turn our attention to DNA repair and the maintenance of genomic stability. This is a critical domain for understanding cellular physiology, disease pathogenesis, and notably, cancer biology. ## The Paramount Importance of Genomic Stability Genomic stability is essential for the accurate inheritance of genetic information across generations and for the proper functioning of individual cells within an organism. DNA uniquely serves as the repository of heritable information and, unlike other biological macromolecules such as RNA, proteins, lipids, and polysaccharides that undergo metabolic turnover, DNA is actively maintained and repaired to preserve its integrity rather than being replaced. ## DNA Repair Defects and Disease: Focus on Cancer Dysfunction in DNA repair mechanisms is implicated in a spectrum of diseases, with cancer being the most prominent. A thorough understanding of DNA repair processes is therefore indispensable for elucidating cancer development, progression, and therapeutic interventions. ## Ubiquitous DNA Damage and Cellular Repair Systems Cells are continuously subjected to DNA-damaging agents originating from both internal metabolic activities (endogenous sources) and external environmental exposures (exogenous sources). To counteract the constant threat of DNA damage, cells have evolved a complex and sophisticated network of DNA repair pathways. These pathways are crucial for preserving the integrity of the genome and preventing the accumulation of mutations that can lead to cellular dysfunction and disease. # Fundamental Principles of DNA Repair ## DNA Integrity and Hereditary Information DNA serves as the fundamental repository of hereditary information, making its structural and informational integrity paramount for life. The substantial energetic investment cells make in DNA repair mechanisms underscores the critical importance of maintaining the genome. ## Energetic Investment in DNA Repair Systems The complexity and scope of DNA repair are reflected in the large number of proteins---hundreds---dedicated to these processes. This extensive molecular machinery represents a significant energetic commitment by the cell, comparable to the energy expenditure for essential processes like transcription and translation. This high energy cost highlights the indispensable role of DNA repair in ensuring cell survival and genomic fidelity. ## Distinction Between DNA Lesions and Mutations ::: definition **Definition 1** (DNA Lesion). *A DNA lesion is a physical or chemical alteration to the DNA molecule. This alteration can be a base modification, strand break, or other forms of damage.* ::: ::: definition **Definition 2** (Mutation). *A DNA lesion becomes a mutation only when it is not repaired before DNA replication and results in a permanent change in the DNA sequence that is heritable in subsequent generations. Most DNA lesions are repaired before replication can fix them into mutations.* ::: Understanding this distinction is essential, as not all DNA damage events lead to permanent genetic changes. ## General Principles of DNA Repair Pathways Several overarching principles govern the operation of DNA repair pathways, ensuring their effectiveness and fidelity. ### Specificity of Lesion Recognition DNA repair pathways exhibit remarkable specificity. Different pathways have evolved to recognize and repair distinct types of DNA lesions. This specialization reflects the diverse nature of DNA damage caused by various endogenous and exogenous agents. ### Pathway Redundancy and Interconnection Redundancy is a key feature of DNA repair systems. Multiple pathways may exist to repair the same type of DNA damage, and enzymes can participate in more than one repair pathway. Furthermore, repair intermediates generated by one pathway can serve as substrates for other pathways. This interconnected and redundant network ensures robust and efficient DNA repair, maximizing the chances of correcting DNA damage. For instance, pyrimidine dimers can be processed by both error-prone translesion synthesis and the high-fidelity Nucleotide Excision Repair (NER) pathway. ### Utilization of Complementary Strand Information The majority of DNA repair pathways leverage the information encoded in the undamaged complementary DNA strand to guide accurate repair. The double-helix structure of DNA, with its inherent base complementarity, is thus fundamental to high-fidelity DNA repair. The undamaged strand serves as a template to restore the correct sequence in the damaged strand. ## Frequency and Efficiency of DNA Repair in Human Cells Human cells are constantly challenged by a high frequency of DNA lesions. It is estimated that each cell in the human body sustains between $10^4$ and $10^6$ DNA lesions per day due to normal metabolic processes and environmental exposures. This translates to an astonishing $10^{18}$ to $10^{20}$ DNA repair events occurring daily throughout the human body. Despite this substantial burden of DNA damage, cellular repair mechanisms are remarkably efficient. Less than one in every 1000 DNA lesions, on average, escapes repair and becomes a permanent mutation. This high efficiency of DNA repair is crucial for maintaining genomic stability and preventing disease. ## DNA Damage Tolerance vs. Repair Mechanisms Cells employ a combination of DNA repair and damage tolerance mechanisms to manage DNA lesions. ### Error-Prone Translesion Synthesis (TLS) Polymerases A subset of DNA polymerases, known as translesion synthesis (TLS) polymerases (e.g., polymerase $\eta$, $\iota$, $\zeta$, $\kappa$, $\lambda$, $\mu$), are specialized for replicating past DNA lesions that would otherwise stall replicative polymerases. These TLS polymerases typically lack proofreading activity and are error-prone. While they allow DNA replication to proceed in the presence of damage, they often introduce mutations at or near the lesion site. Thus, TLS is considered a DNA damage tolerance mechanism rather than a true repair pathway, as it prioritizes replication completion over maintaining the original DNA sequence. ### Error-Prone Non-Homologous End Joining (NHEJ) Non-Homologous End Joining (NHEJ) is a major pathway for repairing DNA double-strand breaks (DSBs). While NHEJ can efficiently rejoin broken DNA ends, it is considered error-prone because it often involves small insertions or deletions at the repair site. NHEJ directly ligates the DNA ends without using a homologous template, which can lead to loss of genetic information and introduce mutations. Despite its error-prone nature, NHEJ is essential for repairing DSBs, which are particularly hazardous lesions that can lead to chromosomal instability and cell death if left unrepaired. # DNA Repair and Cancer ## Causal Link Between DNA Damage and Cancer A significant majority of cancers, approximately 80%, are etiologically linked to DNA-damaging agents. This strong association underscores the critical role of DNA damage in the initiation and progression of cancer. ## Age-Dependent Cancer Risk and Cumulative DNA Damage The incidence of cancer exhibits an exponential increase with age, rather than a linear progression. This exponential relationship reflects the cumulative impact of DNA damage over an individual's lifespan and the progressive accumulation of mutations through successive cell divisions. Increased age correlates with prolonged exposure to mutagens and a greater number of cell cycles, thereby elevating the probability of mutation fixation and subsequent tumorigenesis. ## Hereditary Cancer Predisposition Syndromes: Defects in DNA Repair Genes Numerous hereditary cancer syndromes are directly attributable to germline mutations in genes encoding components of DNA repair pathways. These inherited mutations compromise DNA repair capacity, predisposing individuals to a heightened risk of developing specific types of cancer. ## Exemplary Cancer Predisposition Syndromes and Defective Repair Pathways Several well-characterized cancer predisposition syndromes serve as compelling examples of the link between DNA repair defects and cancer susceptibility. ### Xeroderma Pigmentosum (XP): Nucleotide Excision Repair (NER) Deficiency Xeroderma Pigmentosum (XP) arises from mutations in genes involved in the Nucleotide Excision Repair (NER) pathway. NER is crucial for repairing bulky DNA lesions, including pyrimidine dimers induced by ultraviolet (UV) radiation. Individuals with XP exhibit extreme sensitivity to sunlight and have a markedly elevated risk of skin cancers due to their inability to efficiently repair UV-induced DNA damage. ### BRCA1/BRCA2-Associated Cancer: Homologous Recombination (HR) Deficiency Mutations in the BRCA1 and BRCA2 genes, key players in the Homologous Recombination (HR) DNA repair pathway, are strongly associated with an increased susceptibility to breast and ovarian cancers, particularly in women. BRCA1 and BRCA2 are essential for the accurate repair of DNA double-strand breaks. The widespread public awareness of BRCA gene mutations has been significantly amplified by personal accounts and preventative health decisions. BRCA1 and BRCA2 mutations are clinically relevant as both prognostic and predictive biomarkers in cancer management. Furthermore, the germline nature of these mutations has implications for genetic counseling and family planning due to the potential for vertical transmission to offspring. ### Hereditary Non-Polyposis Colorectal Cancer (HNPCC) / Lynch Syndrome: Mismatch Repair (MMR) Deficiency Hereditary Non-Polyposis Colorectal Cancer (HNPCC), also known as Lynch Syndrome, is caused by inherited mutations in genes of the Mismatch Repair (MMR) pathway. MMR is critical for correcting base-base mismatches and insertion/deletion loops that arise during DNA replication. Individuals with HNPCC have a pronounced predisposition to colorectal cancer and other gastrointestinal malignancies. The high proliferative rate and rapid turnover of intestinal epithelial cells render them particularly vulnerable to replication errors, highlighting their dependence on functional MMR. ## Driver and Passenger Mutations in Cancer Development Mutations implicated in cancer development are broadly categorized as either driver or passenger mutations, based on their functional roles in tumorigenesis. ::: definition **Definition 3** (Driver Mutations). *Driver mutations, also termed founder mutations, are mutations that directly initiate and drive the process of tumorigenesis. These mutations typically occur early in cancer development and affect genes that are critically involved in regulating cell growth, survival, and genomic stability. Genes encoding DNA repair proteins are frequently targets of founder mutations, as their disruption can lead to genomic instability, a hallmark of cancer.* ::: ::: definition **Definition 4** (Passenger Mutations). *Passenger mutations, in contrast, are mutations that accumulate during tumor progression but do not directly initiate tumorigenesis. However, a subset of passenger mutations, termed progressor mutations or essential passenger mutations, confer selective advantages to tumor cells, contributing to tumor evolution, metastasis, and drug resistance. These mutations often arise as a consequence of the genomic instability initiated by founder mutations, enabling tumor cells to adapt and evolve within the selective pressures of the tumor microenvironment and therapeutic interventions.* ::: ### DNA Repair Genes as Frequent Targets of Founder Mutations Genes responsible for maintaining genomic stability, notably DNA repair genes, are frequently targeted by founder mutations in cancer. Examples of such genes include p53 (a central tumor suppressor involved in DNA damage response and repair), cell cycle regulatory genes (such as CDKN2A), and proto-oncogenes (like K-ras). Mutations in these genes disrupt fundamental cellular control mechanisms, initiating acascade of events that can lead to uncontrolled cell proliferation and tumor development. ## Cellular Origin of Tumors: The Cancer Stem Cell Hypothesis The cancer stem cell hypothesis posits that tumors originate from a subpopulation of cells within the tumor termed cancer stem cells (CSCs). These cells possess stem-cell-like properties, including self-renewal capacity and the ability to differentiate into various cell types within the tumor. CSCs are proposed to be responsible for tumor initiation, sustained tumor growth, metastasis, and recurrence after therapy. ## Genomic Instability as an Enabling Hallmark of Cancer The \"hallmarks of cancer\" represent a conceptual framework describing the essential biological capabilities acquired by cancer cells during tumorigenesis. Genomic instability is now recognized as an enabling hallmark that underpins the acquisition of many other hallmarks. ### Genomic Instability: Fueling Tumor Evolution and Heterogeneity Genomic instability, characterized by an increased rate of mutations and chromosomal aberrations, is not merely a consequence of tumorigenesis but actively facilitates it. In the early stages of tumor development, genomic instability promotes the accumulation of driver mutations that initiate and accelerate tumor evolution. However, while initially promoting genetic diversity and adaptation, excessive genomic instability can be detrimental, leading to cell death or growth arrest. Therefore, cancer cells must strike a balance, maintaining a level of genomic instability that is sufficient to drive evolution and adaptation but not so extreme as to compromise cell viability. ### Interplay of Hallmarks and the Role of Genomic Instability The various hallmarks of cancer are interconnected and operate synergistically to drive tumor development and progression. Genomic instability acts as a critical enabler, facilitating the acquisition and manifestation of other hallmarks, including sustained proliferative signaling, evasion of growth suppressors, resistance to cell death (apoptosis), induction of angiogenesis, and activation of invasion and metastasis. Chronic inflammation, often present in the tumor microenvironment, further contributes to genomic instability by producing reactive oxygen species and inflammatory mediators that can damage DNA, creating a vicious cycle that promotes tumorigenesis. ## Intracellular Signaling Circuits and Key Cancer Genes Intracellular signaling circuits involving proto-oncogenes (e.g., Ras, Myc), tumor suppressor genes (e.g., p53), and cell cycle regulators (e.g., CDKN2A) are frequently dysregulated in cancer cells, contributing to the hallmark capabilities of cancer. ### Key Regulatory Genes in Cancer: p53, Cell Cycle Regulators, Proto-oncogenes - **p53:** A central tumor suppressor gene, p53 is among the most frequently mutated genes in human cancers. Mutant p53 proteins often exhibit oncogenic or oncomorphic gain-of-function activities, further promoting tumorigenesis. - **Cell Cycle Regulators (e.g., CDKN2A):** Genes that control cell cycle progression are frequently mutated or dysregulated in cancer, leading to uncontrolled proliferation. CDKN2A, encoding p16INK4a and p14ARF, is a key tumor suppressor locus often inactivated in various cancers. - **Proto-oncogenes (e.g., Ras, Myc):** Proto-oncogenes, when mutated or overexpressed, can become oncogenes that drive cell proliferation and survival. Ras family GTPases and Myc transcription factors are prominent examples of proto-oncogenes frequently activated in cancer. ### Therapeutic Implications of Targeting Founder Mutations From a therapeutic perspective, targeting founder mutations represents the most effective strategy for achieving durable cancer eradication.However, tumors are often diagnosed at later stages, when progressor mutations and intratumoral heterogeneity are already established. This clonal evolution and heterogeneity pose significant challenges to therapy, as treatments targeting progressor mutations may encounter resistance due to the selective outgrowth of pre-existing resistant subclones within the tumor. A comprehensive understanding of the evolutionary dynamics of tumor development, the temporal order of mutation acquisition, and the distinct roles of founder and progressor mutations is crucial for developing more effective and personalized cancer therapies that can overcome resistance and improve patient outcomes. # Conclusion In summary, this lecture has detailed the elongation, translocation, and termination phases of translation, emphasizing the energy expenditure inherent in protein synthesis and the mechanisms of action of antibiotics that target translation. We then transitioned to an introduction to DNA repair and genomic stability, underscoring the vital role of DNA repair in preventing mutations and cancer. We discussed the fundamental principles of DNA repair, the diverse types of DNA damage, and the established link between deficiencies in DNA repair pathways and cancer predisposition syndromes. We introduced the concepts of driver and passenger mutations in cancer development and the hallmarks of cancer, setting the stage for a more in-depth exploration of specific DNA repair pathways and their functions in maintaining genomic integrity and preventing tumorigenesis in subsequent lectures. The next lecture will delve into the specific types of DNA damage and the detailed mechanisms of various DNA repair pathways.