Chapter 11: Translation

Here is an extensive study guide for Chapter 11, "Translation," drawing on information from the provided sources and our conversation history:

Chapter 11: Translation - Study Guide

Translation is the process of synthesizing a polypeptide from the information in an mRNA molecule. Proteins are made from 20 different amino acids.

11.1: Overview of Translation

• Translation is the synthesis of a polypeptide from the information in an mRNA.

• Proteins are constructed from 20 different amino acids.

• The complex task of protein synthesis is carried out by a large molecular machine called the ribosome.

• Ribosomes are composed of RNA (ribosomal RNA or rRNA) and protein (ribosomal proteins or r-proteins).

• All ribosomes have two subunits: a small subunit and a large subunit.

  • The small subunit is responsible for deciphering the mRNA.

  • The large subunit mediates the formation of peptide bonds between amino acids.

• The mass of the large subunit is about twice that of the small subunit, and the overall mass of the ribosome is roughly two-thirds RNA and one-third protein.

• The overall diameter of a bacterial ribosome is about 220 Å.

• Two essential processes for translation are:

  • Deciphering triplet codons in mRNA.

  • Incorporation of amino acids encoded by the triplets into a growing polypeptide chain.

11.2: tRNA and the Genetic Code

• Transfer RNAs (tRNAs) link the mRNA code to protein sequences.

• tRNA molecules are small RNAs, ranging from 75 to 94 nucleotides in length.

• There are many different tRNAs, each specific to a particular amino acid. This specificity is determined by aminoacyl-tRNA synthetases.

• tRNAs have a cloverleaf structure in two dimensions with four base-paired stems or arms interspersed with single-stranded loops.

  • The acceptor stem at the 5' and 3' ends has a conserved 3' CCA tail, which is the attachment point for amino acids.

  • The anticodon loop contains the anticodon, a three-nucleotide sequence that base-pairs with mRNA codons in an antiparallel fashion.

• The nucleobases of the anticodon (positions 34-36) are typically stacked and follow a U-turn conformation, positioning them for effective base-pairing.

• A hypermodified purine residue often occurs just after the anticodon (position 37), which helps in aligning codon-anticodon base-pairing and ensuring high fidelity. An example is wybutosine (Y) in some eukaryotic tRNAs.

• The DHU (D) loop and TψC (T) loop are named after the modified bases they contain: dihydrouridine (D) and ribothymidine (T) and pseudouridine (ψ).

• About 10% of nucleotides in tRNA are post-transcriptionally modified. These modifications are specific to each tRNA species and are proposed to:

  • Aid in folding.

  • Stabilize overall structure.

  • Increase specificity of interactions with translation system components (including codons).

• The three-dimensional structure of tRNA is an L shape, which appropriately spaces the anticodon and acceptor stems for interactions with aminoacyl-tRNA synthetases and the ribosome.

• The genetic code defines the relationship between mRNA sequences (codons) and protein sequences (amino acids).

• Each triplet mRNA codon specifies either a single amino acid (sense codon) or no amino acid (nonsense codon or stop codon).

• There are three stop codons: UAA, UAG, and UGA.

• Some amino acids are specified by a single codon (e.g., tryptophan, methionine), while others are specified by multiple codons.

• In many cases, the first two nucleotides of a codon are sufficient to specify an amino acid, and the third nucleotide can be variable (this contributes to wobble).

• The first two positions of the codon pair with the third and second positions of the anticodon via strict Watson-Crick pairing.

• Wobble pairing occurs at the third position of the codon and the first position (5' proximal) of the anticodon, allowing certain non-Watson-Crick pairings (e.g., G-U) to be accepted. Inosine (I) in the anticodon can pair with U, C, or A in the mRNA.

• Due to wobble pairing, the number of tRNAs needed to decode all 61 sense codons is less than 61 (generally about 40).

• The genetic code is nearly universal.

• The genetic code has likely evolved to minimize the deleterious effects of mutations, where single nucleotide changes often result in the insertion of chemically similar amino acids.

11.3: Amino-acyl tRNA Synthetases

• Aminoacyl-tRNA synthetases are enzymes that attach the correct amino acid to its cognate tRNA.

• This process, aminoacylation, is highly accurate (fewer than one error per 10⁴ aminoacylation events).

• Each amino acid has its own aminoacyl-tRNA synthetase.

• The specific amino acid with which a tRNA is loaded is indicated with a superscript (e.g., tRNA\textsuperscript{Met}).

• The correct amino acid for a tRNA is referred to as cognate.

• The loading process occurs in two steps:

  1. The amino acid is activated by attachment of AMP, releasing pyrophosphate and providing energy. The resulting aminoacyl-adenylate remains attached to the enzyme.

  2. The enzyme then transfers the amino acid to the 2' or 3' OH of the ribose of the terminal adenosine on the tRNA 3' CCA tail.

• In M. jannaschii, a glutamate tRNA synthetase (GluRS) initially loads glutamate onto tRNA\textsuperscript{Gln}, and then glutamate is converted to glutamine by a transamidase. Similarly, phosphorylated serine is loaded onto a tRNA by SepRS and then converted to cysteine by a cysteine desulfurase. These are exceptions to the general rule.

11.4: Structure of the Ribosome

• The ribosome is a large macromolecular machine that facilitates the interaction between tRNAs and mRNA and forms peptide bonds.

• It ranges in mass from 2.5 MDa in bacteria to more than 4 MDa in eukaryotes.

• Approximately two-thirds of the ribosome's mass is rRNA, and about one-third is ribosomal proteins.

• Ribosomes are highly conserved across all life forms in structure and function.

• All ribosomes have a large subunit and a small subunit.

  • The small subunit mediates interactions between mRNA and tRNA.

  • The large subunit catalyzes peptide bond formation.

• These events are integrated at the interface between the two subunits, where movements can shift the tRNA-mRNA complex as amino acids are added.

• The ribosome has an exit tunnel in the large subunit through which the polypeptide is extruded.

• The interface between subunits is rich in rRNA elements and relatively poor in proteins, while ribosomal proteins are more evenly distributed on the exterior.

• Some ribosomal proteins have globular domains on the exterior with long arms extending into the rRNA structure, acting to compact the rRNA.

• Eukaryotic ribosomes are larger and more complex than bacterial ribosomes, with additional layers of RNA and protein. Archaea have an additional protein layer, yeast more protein and RNA, and human ribosomes have RNA expansion segments. These additions likely facilitate more complex translational regulation.

• The large rRNAs are divided into domains based on secondary structure.

  • The 16S (18S in eukaryotes) rRNA has three major and one minor domain.

  • The 23S (28S in eukaryotes) rRNA has six domains.

• In the small subunit, rRNA domains fold independently, while in the large subunit, they are interwoven. This organization may reflect functional differences.

• In addition to the large rRNAs, the large subunit in bacteria and eukaryotes has a 5S RNA. Eukaryotes also have a 5.8S rRNA, which is derived from the 5' end of the large subunit rRNA.

• rRNAs are central to ribosomal functions, reflected in their highly conserved structure, including primary sequence in certain stretches. Many ribosomal proteins are also highly conserved.

• Ribosomes have three binding sites for tRNAs: the aminoacyl (A) site, the peptidyl (P) site, and the exit (E) site.

• The small ribosomal subunit binds the mRNA and facilitates mRNA codon-aminoacyl-tRNA anticodon interactions.

• The large ribosomal subunit catalyzes peptide bond formation.

• The mRNA is threaded through the ribosome in the 5' to 3' direction, and the polypeptide is synthesized from the N-terminus to the C-terminus, exiting through the tunnel.

• During translation, aminoacyl-tRNA binds to the A site, then moves to the P site after peptide bond formation, and finally the uncharged tRNA moves to the E site before leaving.

• Complementary base-pairing between tRNA and mRNA is required in the A and P sites (possibly not the E site).

11.5: The Translation Cycle: the Ribosome in Action

• Translation involves four basic steps: initiation, elongation, termination, and ribosome recycling.

• Each step is facilitated by protein factors that ensure high accuracy and efficiency.

• During initiation, the AUG start codon is identified by the ribosome, a specialized initiator methionine tRNA, and initiation factors (IFs or eIFs). This generates a ribosome with Met-tRNA in the P site.

• During elongation, amino acids are sequentially added to the growing polypeptide chain as the ribosome moves along the mRNA (5' to 3'). This cycle has three steps:

  1. Decoding: The mRNA codon in the A site specifies the incoming aminoacyl-tRNA, which is loaded with the help of elongation factors (EFTu/eEF1A) and GTP hydrolysis. These factors also "evaluate" the codon-anticodon interaction for a match.

  2. Peptide bond formation: The peptidyl transfer center of the large subunit catalyzes the joining of the new amino acid to the growing polypeptide chain, which is transferred from the tRNA in the P site to the tRNA in the A site. The energy comes from ATP used in amino acid activation.

  3. Translocation: Another elongation factor (EFG/eEF2) promotes the movement of the mRNA-tRNA complex by three nucleotides through the ribosome, moving the peptidyl-tRNA to the P site and the next codon to the A site. This requires GTP hydrolysis.

• Termination occurs when the ribosome reaches a stop codon in the mRNA.

• Ribosome recycling involves the disassembly of the ribosome, mRNA, and remaining tRNA after protein release.

• The hybrid states model suggests that tRNAs ratchet through the ribosome, maintaining contact with one subunit while moving relative to the other. They move first with respect to the large subunit after peptide bond formation, and then EFG/eEF2 promotes movement of the anticodon ends with respect to the small subunit. This results in "hybrid states" where tRNAs straddle different binding sites on the subunits, and the ribosome is in a "rotated" state.

11.6: Protein Factors Critical to the Translation Cycle

• The translation cycle is facilitated by protein factors that increase the speed, precision, and processivity of translation.

• These factors include initiation factors (IFs/eIFs), elongation factors (EFs/eEFs), and release factors (RFs/eRFs).

• Many of these factors are GTPases that cycle between GTP-bound (active) and GDP-bound (inactive) states. These transitions are typically promoted by GTPase-activating proteins (GAPs) and guanine-nucleotide exchange factors (GEFs).

  • Bacterial EFTs (related to eukaryotic eEF1Bα) is the GEF for EFTu/eEF1A.

  • Eukaryotic eIF5 is the GAP for eIF2 (involved in initiator tRNA loading).

  • The ribosome itself can act as a GAP (e.g., for EFTu upon cognate tRNA binding).

• GTPases involved in translation likely interact with the flexible stalk region of the large ribosomal subunit and communicate with the ribosome's interior via protein domains, associated factors, or tRNA substrates, leading to functional consequences like translocation or tRNA loading.

• Protein factors are not absolutely required for minimal in vitro translation on simple mRNAs, but they significantly enhance the process in vivo.

11.7: Translation Initiation – Shared Features in Bacteria and Eukaryotes

• Translation initiation brings together the ribosome, mRNA, and the initiator tRNA to the start codon (AUG).

• The initiator tRNA, carrying methionine, binds to the P site of the small ribosomal subunit.

• A GTPase initiation factor (IF2 in bacteria, eIF2 and eIF5B in eukaryotes) likely plays a role in directing the binding of the initiator tRNA to the P site.

• The process differs significantly between bacteria and eukaryotes.

11.8: Bacterial Translation Initiation

• Locating the AUG start site in bacteria is relatively simple.

• Bacterial mRNAs are often polycistronic, containing multiple open reading frames (ORFs), each encoding a different protein with its own initiation and termination codon.

• Initiation codons (typically AUG, sometimes GUG) are usually preceded by a Shine-Dalgarno sequence (or ribosome-binding site), a polypurine tract located 6-8 nucleotides upstream of the AUG.

• The Shine-Dalgarno sequence (consensus AGGAGGU) base-pairs with a polypyrimidine tract at the 3' end of the bacterial 16S rRNA (the anti-Shine-Dalgarno sequence) in the small ribosomal subunit.

• The strength of this interaction, influenced by deviations from the consensus sequence, affects translation efficiency.

• This base-pairing guides the AUG into the P site of the ribosome for interaction with the initiator f-Met-tRNA\textsuperscript{fMet}.

• Some bacterial and archaeal mRNAs lack a leader sequence upstream of the AUG and use different mechanisms for leaderless initiation.

• Initiator tRNA binding is guided by a small set of protein initiation factors (IF1, IF2, IF3) in bacteria.

11.9: Eukaryotic Translation Initiation

• Eukaryotic initiation is more complex than in bacteria.

• It also occurs at an AUG codon decoded by a special initiator Met-tRNAiMet (distinct from bacterial f-Met-tRNA\textsuperscript{fMet}).

• Eukaryotic ribosomes identify the AUG start site using a scanning mechanism that begins at the 5' cap structure of the mRNA.

• The eukaryotic 40S small ribosomal subunit does not bind directly to the mRNA upstream of the AUG using a Shine-Dalgarno-like sequence because the 18S rRNA lacks the anti-Shine-Dalgarno tract.

• Exceptions include internal ribosome entry sites (IRESs) where 40S subunits can bind directly near the AUG.

• Eukaryotic mammalian initiation requires at least 11 distinct initiation factors (eIFs), totaling around 28 polypeptides. Bacterial initiation only requires three.

• Eukaryotic mRNAs have unique features aiding initiation: the 7-methyl guanosine cap at the 5' end and the poly(A) tail at the 3' end.

• These features allow for preparatory steps involving the eIF4 family of factors, priming the mRNA for scanning.

• The 5' cap is bound by eIF4E (cap-binding protein), and the 3' poly(A) tail is bound by poly(A) binding protein (PABP).

• These proteins interact with eIF4A/4B (ATPase for unwinding mRNA structure) and eIF4G (scaffold protein), leading to the formation of a closed loop complex on the mRNA, which enhances translation efficiency and acts as a quality control feature.

• Scanning begins with the 48S preinitiation complex (40S subunit bound to Met-tRNAiMet-eIF2-GTP and other eIFs) binding to the eIF4 factor-bound mRNA via eIF3 interaction with eIF4G.

• eIF4A unwinds secondary structures during scanning, and the initiator tRNA evaluates codons.

• Upon encountering a suitable AUG codon (influenced by the Kozak consensus sequence and eIF1), eIF5 acts as a GAP, triggering GTP hydrolysis on eIF2. This is a fidelity checkpoint.

• After start site recognition, initiation factors dissociate, and the 60S large subunit joins, a step promoted by the GTPase eIF5B (eukaryotic homolog of IF2) and GTP hydrolysis.

• The completed 80S initiation complex is now ready for elongation.

11.10: Translation Elongation: Decoding, Peptide Bond Formation and Translocation

• Elongation follows initiation, with the ribosome poised on the mRNA with initiator Met-tRNAiMet in the P site and an empty A site.

• The elongation cycle is highly conserved between bacteria and eukaryotes.

• It consists of three steps: decoding, peptide bond formation, and translocation.

  1. Decoding: The ribosome selects an aminoacyl-tRNA with an anticodon complementary to the mRNA codon in the A site. This is facilitated by EFTu-GTP (bacteria) or eEF1A-GTP (eukaryotes), which escorts the aminoacyl-tRNA to the ribosome and ensures high fidelity. EFTu/eEF1A uses GTP hydrolysis to "evaluate" the codon-anticodon interaction and deposit the correct tRNA. The accuracy is around 10⁻³ to 10⁻⁶ errors per codon. Cognate tRNAs bind more tightly and are subject to "initial selection" and "proofreading" before and after GTP hydrolysis. The decoding center in the small subunit (involving universally conserved rRNA nucleotides like A1492, A1493, and G530 in 16S rRNA) recognizes the geometry of the cognate codon-anticodon helix, inducing conformational changes that stimulate GTPase activation on EFTu/eEF1A and accommodation of the tRNA into the A site.

  2. Peptide bond formation: Once the correct aminoacyl-tRNA is in the A site and peptidyl-tRNA is in the P site, the peptidyl transferase center in the large ribosomal subunit catalyzes peptide bond formation. This active site is primarily made of rRNA. The growing polypeptide chain is transferred to the amino acid on the tRNA in the A site. The energy for this comes from aminoacyl-tRNA activation by synthetases.

  3. Translocation: EFG (bacteria) or eEF2 (eukaryotes), using energy from GTP hydrolysis, promotes the movement of the mRNA by three nucleotides relative to the ribosome. This shifts the peptidyl-tRNA from the A site to the P site, the deacylated tRNA from the P site to the E site (for exit), and brings the next codon into the A site.

• The ribosome continues this elongation cycle, gliding along the mRNA and extending the polypeptide chain.

11.11: Translation Termination, Recycling and Reinitiation

• Elongation continues until the ribosome encounters a stop codon (UAA, UAG, or UGA) in the A site.

• These stop codons are recognized by class 1 release factors (RFs), not tRNAs.

  • In bacteria: RF1 (recognizes UAA, UAG) and RF2 (recognizes UAA, UGA), showing overlapping specificity.

  • In eukaryotes: eRF1 recognizes all three stop codons.

• Release factors promote hydrolysis of the bond between the completed polypeptide chain and the peptidyl-tRNA in the P site, releasing the protein.

• Class 1 release factors are bifunctional, linking codon recognition in the small subunit with catalysis in the large subunit, and their structure mimics tRNAs.

• Class 2 release factors (GTPases) are also important for termination.

  • In bacteria: RF3 (related to EFG) appears to function after peptide release to promote dissociation of the class 1 release factor, coupled to GTP hydrolysis.

  • In eukaryotes: eRF3 (related to EFTu/eEF1A) seems to escort eRF1 and uses GTP hydrolysis to promote its own departure after peptide release.

• In eukaryotes, after eRF3 departs, an AAA+ ATPase ABCE1 (or Rli1 in yeast) binds and promotes peptide release even without ATP hydrolysis. eRF1 remains bound after peptide release.

• Ribosome recycling follows peptide release, disassembling the ribosome complex.

  • In bacteria: After RF dissociation (assisted by RF3), ribosome recycling factor (RRF) wedges between subunits and, with EFG and GTP hydrolysis, promotes disassembly. IF3 then binds to the small subunit to stabilize the dissociated state.

  • In eukaryotes: eRF1 remains bound and helps promote subunit dissociation, enhanced by ABCE1 and ATP hydrolysis. Core initiation factors (eIF1, 1A, 3) then trap the dissociated subunits. There is no RRF homolog in eukaryotes.

• Reinitiation of translation on downstream ORFs in polycistronic mRNAs is common in bacteria. Each ORF typically has its own Shine-Dalgarno sequence. Inefficient ribosome recycling can lead to the small subunit remaining associated with the mRNA and scanning for another AUG. Eukaryotic genes are mostly monocistronic, but some have upstream ORFs (uORFs) that can regulate gene expression via reinitiation efficiency.

11.12: Ribosome Rescue in Bacteria and Eukaryotes

• The Problem of Arrested Ribosomes: Normally, ribosomes complete translation, terminate, and are recycled. However, various events can cause ribosomes to pause, stall, or become arrested on the mRNA template.

• Cellular Responses to Arrested Ribosomes: Cells have mechanisms to deal with arrested ribosomes, which generally include:

  • Targeting the mRNA for decay.

  • Targeting the incomplete polypeptide for proteolysis.

  • Recycling the stalled ribosome for future translation.

  • Ribosome Rescue in Bacteria:

    • Truncated mRNAs are a threat: If a ribosome reaches the physical end of an mRNA without encountering a stop codon, it becomes trapped and unavailable for further translation, potentially producing a truncated polypeptide.

    • tmRNA protein tagging system: Bacteria use a unique RNA molecule called tmRNA to rescue ribosomes stalled on truncated mRNAs.

    • Dual function of tmRNA: tmRNA acts first like a tRNA by adding an alanine residue to the growing peptide chain. Then, it acts as an mRNA by encoding an additional ten amino acids followed by a stop codon.

    • Degradation tag: The ten added amino acids form a degradation tag, which signals cellular proteases to degrade the truncated protein.

    • Mechanism of tmRNA action: The tmRNA-SmpB complex (a protein partner) binds to the ribosomal A site, mimicking a tRNA. SmpB monitors the length of mRNA in the ribosome to selectively target stalled ribosomes on truncated mRNAs.

    • Completion of rescue: After the tag is added, the ribosome reaches the tmRNA-encoded stop codon, leading to standard termination and ribosome recycling.

  • Ribosome Rescue in Eukaryotes:

    • Full-length mRNAs are typically translated: Eukaryotic translation initiation strongly depends on the 5' cap and 3' poly(A) tail, so truncated mRNAs are less likely to be efficiently translated.

    • Causes of ribosome pausing/stalling: Ribosomal arrest can occur due to significant mRNA structure, specific peptide stalling sequences, or low abundance of a required aminoacyl-tRNA.

    • No-go decay (NGD) pathway: Stalling events can trigger NGD, leading to degradation of the mRNA and incomplete protein (via the ubiquitin-proteasome system) and ribosome rescue.

    • Factors involved in NGD: Homologs of termination factors, Pelota (or Dom34 in yeast) and Hbs1 (related to eRF1 and eRF3, respectively), promote a recycling reaction that dissociates the ribosomal subunits, releasing the mRNA and peptidyl-tRNA.

    • Non-stop decay (NSD) pathway: Occurs when an mRNA lacks a stop codon or is truncated before it, and the ribosome reads into the poly(A) tail.

    • Factors involved in NSD: Similar to NGD, Pelota (Dom34) and Hbs1, along with another eRF3 homolog Ski7 (which also interacts with the exosome complex), are likely involved in rescuing ribosomes in NSD.

    • Other potential factors: Other factors are likely involved in detecting stalled ribosomes and targeting the mRNA and truncated protein for degradation.

  • Nonsense-Mediated Decay (NMD) - A Related but Distinct Process:

    • NMD is an mRNA surveillance pathway that targets mRNAs with premature termination codons (PTCs) for degradation.

    • In higher eukaryotes, PTCs are often distinguished from normal stop codons by their location upstream of exon junction complexes (EJCs) left by the splicing machinery.

    • Factors like Upf1, Upf2, and Upf3 are involved in NMD, bridging the ribosome complex and the EJC.

    • NMD leads to mRNA and potentially protein degradation and ribosome rescue, but it's triggered by premature stop codons within the ORF, not necessarily stalled ribosomes at the end of an mRNA or due to other impediments.

11.13: Recoding: Programmed Stop Codon Read-Through and Frameshifting

• This section discusses how biological systems can "flex" the genetic code.

• Recoding refers to exceptions to the standard decoding rules, such as programmed stop codon read-through (where the ribosome bypasses a stop codon and continues translation) and frameshifting (where the ribosome shifts the reading frame), leading to different protein products from the same mRNA.

11.14: Antibiotics that Target the Ribosome

• Many antibiotics disrupt translation in bacteria and fungi by targeting the ribosome or translation factors. They can be bactericidal (kill) or bacteriostatic/fungistatic (inhibit growth).

• Antibiotics often exploit subtle structural differences between bacterial/fungal and mammalian ribosomes.

• Some antibiotics bind directly to the ribosome, while others interact with translation factors.

• Due to their small size compared to the ribosome, antibiotics must target functionally critical regions.

• Erythromycin (a macrolide) binds in the exit tunnel of the large subunit, blocking the growing peptide chain.

• Chloramphenicol binds centrally in the active site of the large subunit, blocking access to nucleotides important for tRNA binding and catalysis.

• Paromomycin (an aminoglycoside) can affect decoding, termination, and recycling, suggesting functionally critical regions might be close together or target movements common to multiple steps.

• Antibiotic resistance mutations often occur in ribosomal components, providing insights into antibiotic mechanisms and ribosome function.

• Streptomycin acts as a miscoding agent by binding to the small subunit and inducing conformational changes with near-cognate tRNAs, leading to incorrect amino acid incorporation. Resistance can arise from mutations in the small subunit protein S12, located near the streptomycin binding pocket. These mutations can increase the ribosome's inherent fidelity, sometimes leading to slower growth in the absence of streptomycin. Additional mutations in S4 and S5 can counteract this effect but cause error-prone translation.

• Antibiotic resistance mutations are more often found in ribosomal proteins than rRNA because bacteria typically have multiple copies of rRNA genes, requiring mutations in a significant fraction for resistance.

• An exception is resistance to macrolides like erythromycin, where the most common mechanism is the acquisition of a methylase gene that modifies the antibiotic binding site (e.g., methylation of A2058 in the large subunit rRNA), preventing binding. Resistance can also occur through direct mutation of the same nucleotide in rRNA genes.