Test 3 (LAST RUTTY TEST!!!)

Reading List:

• Chapter 9: not 9.4 and not the section on yeast and mating

• Chapter 10: not 10.2-10.3, 10.8 and 10.11

• Chapter 11: ALL

• Chapter 12: ALL

Chapter 8: Transcription

• Overview of Transcription:

  • The information stored in DNA is used to make a functional protein or RNA molecule.

  • Transcription is the copying of one strand of DNA (the coding strand) into an RNA molecule (a transcript).

  • RNA polymerase replicates the coding strand of DNA by using the complementary strand or the template strand as a template.

  • The RNA sequence is therefore identical to the coding strand, synthesized in the 5’ to 3’ direction.

  • Transcription is heavily regulated to produce only the required RNA at the right times.

  • Chromatin-packaged DNA presents a challenge to transcription, as nucleosomes can prevent transcription machinery from binding to DNA.

  • Eukaryotic transcription requires additional protein complexes.

• Start of bacterial transcription:

  • A protein termed a sigma factor binds to the -10 and -35 regions of bacterial promoters.

  • Sigma factors bind sequences that define bacterial promoters.

  • Bacterial promoters generally have two elements: the -35 element and the -10 element, roughly 35 and 10 bases upstream of the transcription start site.

  • Each sigma factor has a preferred binding sequence and preferred spacing between the -35 and -10 elements.

  • This preference regulates transcription; sequences and spacing closer to a sigma factor's preference result in tighter binding and higher transcription rates.

  • Some sigma factors are regulated in response to environmental or developmental cues.

  • Bacterial RNA polymerase is a large complex, with the core enzyme composed of β, β’, two α, and ω subunits.

  • The active site is located at the base of the cleft formed by the β and β’ subunits.

  • The α subunit has N-terminal (αNTD) and C-terminal (αCTD) domains connected by a flexible linker, capable of interacting with the DNA template and stabilizing the enzyme during high gene expression.

  • RNA polymerases share five central subunits with conserved amino acid sequences and 3D structures, especially at the active site.

• Signals in DNA Tell RNA Polymerase Where to Start and Finish Transcription:

  • Transcription starts in the promoter region.

  • Sigma factors bind to promoter elements and asymmetrically, determining the transcription direction.

  • Transcription terminates when the RNA polymerase encounters a terminator sequence in the DNA, causing the polymerase to release the RNA transcript and dissociate from the DNA.

• Eukaryotic RNA Polymerase Requires General Transcription Factors:

  • Eukaryotic transcription requires general transcription factors, many different proteins that assemble at the promoter.

  • TFIID, containing the TATA-binding protein (TBP), binds to the TATA box and distorts the DNA.

  • TFIIB then binds, positioned asymmetrically and resembling bacterial sigma factor.

  • After TFIID and TFIIB, TFIIA binds to stabilize the TBP-DNA interactions, followed by TFIIE and TFIIH (TFIIH uses ATP to unwind DNA).

  • Eukaryotic RNA polymerases (Pol I, Pol II, Pol III) have similar core subunits and all use TBP for initiation, making TBP part of different complexes for each polymerase.

  • Another large complex, the Mediator, with over 20 subunits, is needed to activate many Pol II transcribed genes.

• Eukaryotic Genes Are Controlled by Combinations of Transcription Regulators:

  • Gene regulation is combinatorial, with multiple control elements and transcription factors acting together to establish patterns of gene expression in different cell types.

  • Different cell types possess different sets of transcription factors.

• The Arrangement of Chromosomes into Looped Domains Keeps Enhancers in Check:

  • Animal and plant chromosomes are arranged in DNA loops, held by specialized proteins, favoring the association of each gene with its proper enhancer.

  • These loops, sometimes called topological associated domains (TADs), range from thousands to millions of nucleotide pairs and are larger than loops between regulatory sequences and promoters.

• Factors Act in Combination:

  • The modular construction of gene regulatory regions, like that of the Eve gene, allows for complex expression patterns.

• Locus control regions (LCRs) are found in more complex eukaryotes:

  • LCRs are important for the coordinated expression of gene families, such as the β-globin gene family.

• Reporter Genes Allow Specific Proteins to Be Tracked in Living Cells:

  • Reporter genes can be used to study the regulation of gene expression by fusing their regulatory regions to a coding sequence for an easily detectable protein.

Chapter 9: Transcriptional Regulation PART I

• Principles of Transcription Regulation:

  • Gene regulation is fundamental for controlling gene expression.

• DNA-Binding Domains in Proteins that Regulate Transcription:

  • Transcription regulatory proteins bind to specific DNA sequences through various DNA-binding domains.

• Mechanisms for Regulating Transcription Initiation in Bacteria:

  • Transcription initiation in bacteria is a key control point, often regulated by the binding of activator or repressor proteins to DNA sequences near the promoter.

  • Competition between regulatory proteins, like the λ phage repressors cI and Cro, can determine the fate of transcription.

Chapter 10: RNA Processing

• Overview of RNA Processing:

  • RNAs synthesized from DNA templates often require processing to become functional.

  • RNA processing includes various modifications summarized in Figure 10.1, such as removal of ends, RNA splicing (removal of introns and joining of exons), 5' capping, 3' polyadenylation, RNA editing (insertion, deletion, or chemical modification of bases), and RNA degradation.

  • RNA processing is required to produce functional RNAs and provides benefits such as regulation of gene expression, generation of diverse RNAs from a single gene (e.g., alternative splicing in the Drosophila dscam gene), and quality control by degrading defective RNAs.

  • Most RNA processing steps are performed by multicomponent macromolecular machines, often ribonucleoproteins (RNPs), which can interact and are localized to specific cellular compartments.

  • RNA processing events are points for regulation and quality control, and are sources of diversity.

  • Fundamentally, RNA processing is required to produce functional RNAs.

• Functions of RNAs in RNA processing machines:

  • Many RNA processing machines are ribonucleoproteins (RNPs).

  • The RNA in RNPs can be structural or have catalytic activities (ribozymes).

  • Some RNPs contain guide RNAs that base pair with pre-RNAs and guide the RNP to the correct place for processing.

  • Both ends of eukaryotic RNAs are modified during transcription.

  • End modifications protect mRNAs from nuclease degradation and help with protein interactions.

  • The 5' ends are capped with a 7-methylguanine nucleotide via a 5'-5' triphosphate linkage.

• tRNA and rRNA Processing:

  • Nucleotides in tRNAs and rRNAs are modified in numerous ways after transcription, on the base or the ribose sugar.

  • Modifications can involve addition of small groups (e.g., methyl) or larger groups (e.g., amino acids).

  • Specific positions in different tRNA and rRNA species are specifically modified, often required for optimal survival and growth.

  • More than 80 different modifications have been seen in tRNAs, contributing to structural stability and interactions with other molecules.

  • Chemical diversity from modifications may allow for higher-order functions, such as proper interaction with mRNA during protein synthesis.

  • Some complex modifications require multiple enzymes.

  • snoRNAs (small nucleolar RNAs) guide methylation and pseudouridylation of eukaryotic and archaeal rRNAs and some tRNAs.

  • snoRNAs associate with proteins to form snoRNPs in the nucleolus.

  • snoRNAs base-pair with target RNAs to direct modification enzymes to specific sites.

  • Most vertebrate snoRNAs are processed from the introns of precursor mRNAs.

  • There are two main classes of snoRNAs: box C/D snoRNAs direct ribose methylation, and box H/ACA snoRNAs direct pseudouridylation.

• mRNA Capping and Polyadenylation:

  • Eukaryotic mRNAs are capped at the 5' end in a multistep process cotranscriptionally by RNA polymerase II.

  • The 5' cap (7-methylguanine linked 5'-5') is essential for efficient transcription elongation and termination, mRNA processing, nuclear export, translation initiation, and protection from 5' exonucleases.

  • Capping occurs soon after RNA polymerase II synthesizes about 20-30 nucleotides, involving removal of a phosphate, addition of GMP, and methylation of guanine.

  • Eukaryotic mRNAs (except metazoan histone mRNAs) are polyadenylated at the 3' end by the addition of a poly(A) tail (around 200 adenosines).

  • The poly(A) tail also protects mRNA from exonuclease degradation and plays a role in translation initiation.

  • Alternative polyadenylation sites (APAs) can lead to different 3' UTR lengths, affecting mRNA stability and translation by altering binding sites for regulatory factors.

  • Polyadenylation involves cleavage of the mRNA after a CA nucleotide between a conserved AAUAAA hexamer and a U/GU-rich region, followed by the addition of the poly(A) tail by poly(A) polymerase.

  • Capping and polyadenylation are intimately coupled to each other and to transcription by the CTD of RNA polymerase II.

  • The CTD interacts sequentially with different processing complexes.

  • RNA polymerases I and III lack a CTD and their transcripts are not capped and polyadenylated.

• RNA Splicing:

  • The coding sequences of many genes, especially eukaryotic mRNAs, are interrupted by non-coding sequences called introns, which must be removed to generate functional RNAs; the coding segments are exons.

  • The removal of introns and joining of exons is RNA splicing.

  • mRNAs, rRNAs, and tRNAs can contain introns.

  • There are different classes of introns with varying mechanisms of excision: some by proteins, some by RNPs, and some are self-splicing, all involving transesterification reactions.

  • Most introns are degraded after removal, but some (e.g., snoRNAs, some miRNAs) can contain functional RNAs.

  • Introns are more common in eukaryotes than bacteria; their existence has allowed for exon shuffling, contributing to the evolution of eukaryotic genomes.

  • Differential removal of introns (alternative splicing) can produce different spliced RNAs from a single gene, increasing the number of gene products.

  • There are three main groupings of introns: Group I introns, Group II introns, and spliceosomal introns.

• Group I introns:

  • Found in bacteria, viruses, lower eukaryotes, and plants.

  • Many are self-splicing ribozymes (~120-450 nucleotides long) that can excise themselves via two sequential transesterifications.

  • The first reaction involves a free guanosine attacking the 5' splice site.

  • The second reaction involves the released 5' exon end attacking the 3' splice site, joining the exons.

  • Splice sites are defined by the 3D structure of the intron and a conserved G-U wobble pair.

  • Some Group I introns require assistance from proteins.

• Group II introns:

  • Found in bacteria and organellar genes of plants and fungi (~400-1000 nucleotides long).

  • The 2' OH of a specific A within the intron attacks the exon1-intron junction.

  • Once released, the intron forms a branched lariat intermediate.

  • Splice sites are determined by the 3D structure of the intron.

  • Some Group II introns are self-splicing, while others need protein cofactors.

  • Mechanistically similar to spliceosomal introns, suggesting a common evolutionary origin.

  • Can act as mobile genetic elements.

  • Often contain open reading frames encoding 'maturase' proteins that aid in splicing and intron mobility.

• Eukaryotic mRNA Splicing by the Spliceosome:

  • Most eukaryotic introns are not self-splicing; splicing is mediated by the spliceosome, a large RNP machine.

  • Eukaryote genes often have several introns that can be very long (thousands of bases), accounting for up to ~90% of a pre-mRNA (e.g., human dystrophin gene with 78 introns).

  • Some eukaryotes (e.g., yeast) have fewer, shorter introns.

  • Spliceosome-catalyzed splicing is similar to that of Group II introns, involving the formation of a lariat intermediate.

  • The spliceosome is composed of five small nuclear ribonucleoproteins (snRNPs: U1, U2, U4, U5, U6), each with a snRNA (100-300 nucleotides) and proteins.

  • snRNAs form base pairs with the pre-mRNA and act as the recognition part of the snRNP.

  • Spliceosome must identify splice sites (5' and 3' splice sites, branch-point nucleotide, polypyrimidine tract) by recognizing short sequence motifs.

  • U1 snRNP recognizes the 5' splice site.

  • Branch-point-binding protein (BBP) recognizes the branch-point sequence.

  • U2AF recognizes the polypyrimidine tract and 3' splice site.

  • The rest of the spliceosome assembles, leading to structural rearrangements and the two transesterification steps of splicing.

  • snRNAs (U2 and U6) are thought to play a central role in catalysis.

  • Protein factors (e.g., PRP8, ATPases, SR proteins, hnRNPs) contribute to spliceosome assembly, fidelity, regulation, and coordination with other mRNA biogenesis processes.

  • The exon junction complex (EJC) is left behind at splice junctions, marking the transcript for export and translation quality control.

  • A minor spliceosome (U12-dependent or AT-AC spliceosome) processes a small subset of introns with unusual splice sites.

  • Trans splicing joins exons from two separate RNA transcripts, observed in nematodes and trypanosomes, where a spliced leader RNA (SL RNA) is joined to various mRNA transcripts.

• Exon Definition and Alternative Splicing:

  • At least 75% of genes in higher eukaryotes undergo alternative splicing, producing different mature mRNAs by splicing different combinations of exons.

  • This leads to diversity in gene expression depending on cell type, developmental stage, and environmental cues.

  • Outcomes of alternative splicing include exon exclusion/inclusion, alternative 5' or 3' splice site usage.

  • Spliceosomes must distinguish true splice sites from cryptic splice sites (similar sequences occurring by chance).

  • Two models for splice site recognition: exon definition (in mammals) and intron definition.

  • Exon definition: 5' and 3' ends of an exon are brought together by interactions between U1 and U2 complex; exons are marked by SR proteins, and introns by hnRNPs masking cryptic sites. Mutation at a 5' splice site can lead to exon exclusion.

  • Intron definition: Introns are defined by interactions between factors bound at sequential 5' and 3' splice sites.

  • Both models involve co-transcriptional marking of splice sites facilitated by the CTD of RNA polymerase II.

  • Alternative splicing is regulated by cis-acting pre-mRNA sequences (splicing enhancers and silencers in exons and introns) recognized by trans-acting cellular factors (e.g., SR proteins, hnRNPs, PTB).

  • Splicing enhancers (ISE, ESE) promote splicing by binding proteins that increase spliceosome recognition. SR proteins often bind ESEs and are key for exon definition.

  • Splicing silencers (ISS, ESS) inhibit splicing by masking sites or blocking spliceosome assembly, leading to exon omission. PTB is an example of a silencer protein.

  • Regulation of alternative splicing involves a complex interplay of pre-mRNA sequences and protein factors expressed at different levels in different cell types. Mutations can also affect splice site recognition and create new exons.

• RNA Editing:

  • RNA editing involves the insertion, deletion, or chemical modification of individual nucleotides, which can change the amino acid sequence of the encoded protein.

• Degradation of Normal RNAs:

  • RNA molecules are degraded by nucleases into recyclable nucleotides.

  • RNA degradation rates are balanced with transcription rates to regulate RNA concentration.

  • RNA degradation can be promoted or blocked in response to various signals.

  • Example: Iron regulation of transferrin receptor mRNA stability by iron regulatory proteins (IRPs) binding to iron response elements (IREs) in the 3' UTR.

  • Under starvation or stress, cells might degrade a large proportion of RNAs.

  • In bacteria, 'interferases' (toxins with endoribonuclease activity) are involved in regulated RNA degradation under stress.

• Degradation of Foreign and Defective RNAs:

  • Foreign RNAs (e.g., from viruses, transposons) and defective RNAs (due to transcription or processing errors) can be harmful.

  • Eukaryotes use RNA interference (RNAi) to degrade double-stranded foreign RNAs, cleaved by Dicer into short fragments that guide Argonaute proteins to cleave target RNAs.

  • Bacteria and archaea use CRISPR systems, RNA-guided systems to degrade foreign DNA and RNA.

  • Defective mRNAs in eukaryotes (with premature stop codons, no stop codons, or stalling sequences) are recognized and degraded by nonsense-mediated mRNA decay (NMD), non-stop decay (NSD), and no-go decay (NGD), often depending on the EJC.

  • In bacteria, stalled ribosomes on defective mRNAs are recognized by tmRNA, which facilitates translation completion and mRNA degradation.

Chapter 11: Translation

• Overview of Translation:

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

  • Proteins are made from 20 different amino acids.

  • The information in the mRNA (nucleic acid) is translated into protein (amino acids) via transfer RNA molecules (tRNA).

  • tRNAs read the mRNA by base-pairing 3 nucleotides – the region on the tRNA is the anticodon, and on the mRNA it is the codon.

  • tRNAs each carry a specific amino acid at the 3' end, attached by aminoacyl-tRNA synthetases.

  • Amino acids attached to tRNAs are joined together in a chain.

  • Ribosomes carry out translation.

  • Ribosomes have small and large subunits.

  • The translation cycle has four basic stages: initiation, elongation, termination, and ribosome recycling.

  • All steps except peptide bond formation depend on auxiliary protein factors.

  • Three core components of translation: tRNA, aminoacyl-tRNA synthetases, and the ribosome.

• tRNA and the Genetic Code:

  • tRNA molecules are small RNAs (75-94 nucleotides) with a characteristic cloverleaf secondary structure (four stems and three loops) and an L-shaped tertiary structure.

  • The 3' CCA tail is the attachment point for amino acids.

  • The anticodon loop contains the anticodon that base-pairs with mRNA codons.

  • The genetic code is specified by triplet nucleotide codons in mRNA.

  • Each codon specifies a single amino acid (sense codon) or a stop signal (nonsense codon: UAA, UAG, UGA).

  • The code is degenerate; most amino acids are specified by multiple codons.

  • The first two nucleotides of a codon often strictly determine the amino acid, while the third position allows wobble pairing with the anticodon, allowing some tRNAs to recognize multiple codons.

  • The genetic code is nearly universal.

  • Aminoacyl-tRNA synthetases attach the correct amino acid (cognate) to its corresponding tRNA with high accuracy. The resulting molecule is aminoacyl-tRNA.

  • Aminoacyl-tRNA is protected by binding to elongation factor EFTu (bacteria) or eEF1A (eukaryotes).

  • Synthetases recognize specific tRNA identity elements (sequence and structural features) primarily in the anticodon loop and acceptor stem.

  • Synthetases select the correct amino acid in a two-step process, including a proofreading or editing site to hydrolyze mis-activated amino acids.

  • Synthetases are divided into two classes (I and II) with different structures and tRNA binding sites.

• Amino-acyl tRNA Synthetases:

  • (Covered within "tRNA and the Genetic Code")

• Structure of the Ribosome:

  • The ribosome is a large macromolecular machine (2.5-4 MDa) composed of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins).

  • rRNA makes up about two-thirds of the ribosome's mass and is central to its function.

  • Ribosomes are highly conserved across life forms and consist of a large subunit and a small subunit.

  • The small subunit mediates mRNA-tRNA interactions, and the large subunit catalyzes peptide bond formation.

  • The interface between subunits is rich in rRNA and relatively poor in proteins.

  • Ribosomal proteins often have extended arms that interact with the rRNA core.

  • Eukaryotic ribosomes have additional layers of RNA and protein compared to bacteria.

  • rRNAs are divided into domains based on secondary structure: 16S/18S rRNA (small subunit) has 3 major and 1 minor domain; 23S/28S rRNA (large subunit) has 6 domains.

  • Domain organization differs between subunits, suggesting functional specialization.

  • The large subunit also contains 5S rRNA (bacteria and eukaryotes) and 5.8S rRNA (eukaryotes).

  • rRNAs are highly conserved in sequence and structure, reflecting their crucial roles.

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

  • tRNAs bind successively at these sites during translation.

• The Translation Cycle: the Ribosome in Action:

  • The translation cycle involves initiation, elongation, termination, and ribosome recycling.

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

  • Initiation: The ribosome identifies the AUG start codon with the help of initiation factors (IFs) and a specialized initiator methionine tRNA, loading it into the P site. In bacteria, this involves the Shine-Dalgarno sequence on the mRNA base-pairing with the 16S rRNA. Eukaryotes use a scanning mechanism from the 5' cap, involving many more initiation factors (eIFs) and a circular mRNA complex.

  • 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. Aminoacyl-tRNA binding: The next aminoacyl-tRNA (carried by EFTu/eEF1A-GTP) binds to the A site based on codon-anticodon complementarity; GTP hydrolysis leads to its stable binding and release of the elongation factor.

    2. Peptide bond formation: The peptidyl transferase center in the large subunit catalyzes the formation of a peptide bond, transferring the growing polypeptide from the tRNA in the P site to the amino acid on the tRNA in the A site.

    3. Translocation: Elongation factor EFG/eEF2-GTP promotes the movement of the mRNA-tRNA complex by three nucleotides, shifting the peptidyl-tRNA to the P site and the empty tRNA to the E site, opening the A site for the next codon.

  • Termination: Occurs when the ribosome reaches a stop codon (UAA, UAG, UGA) in the mRNA. These are recognized by class 1 release factors (RF1, RF2 in bacteria; eRF1 in eukaryotes), which promote hydrolysis of the bond between the polypeptide and the tRNA in the P site, releasing the completed polypeptide. Class 2 release factors (RF3 in bacteria; eRF3 in eukaryotes), GTPases, are also involved.

  • Ribosome recycling: The ribosome disassembles into its subunits, releasing the remaining tRNA and mRNA, facilitated by ribosome recycling factor (RRF) and EFG in bacteria, and ABCE1/Rli1 and eRF1 in eukaryotes. Initiation factors then bind to the small subunit to prevent premature reassociation.

• Protein Factors Critical to the Translation Cycle:

  • Protein factors facilitate each step of translation (except peptide bond formation), contributing to speed, precision, and processivity.

  • Many are GTPases (G proteins) that use GTP hydrolysis to drive conformational changes linked to ribosome progression (e.g., EFTu/eEF1A, EFG/eEF2, RF3/eRF3, IF2/eIF5B, eIF2).

  • Some factors bind to the ribosome to prevent inappropriate interactions (e.g., IF1, IF3, eIF1, eIF1A).

  • Molecular mimicry is used by some factors; EFG/eEF2 mimics tRNA, and release factors also structurally resemble tRNA.

  • GTPase activity is often regulated by GTPase-activating proteins (GAPs) and guanine-nucleotide exchange factors (GEFs); the ribosome itself can act as a GAP in some cases (e.g., for EFTu/eEF1A).

  • GTPases involved in translation interact with the flexible stalk region of the large ribosomal subunit.

• Translation Initiation – Shared Features in Bacteria and Eukaryotes:

  • Both systems initiate at an AUG codon decoded by a special initiator Met-tRNA.

  • Initiation involves the interaction of the ribosome, the initiator tRNA, and protein initiation factors.

  • Initiation generates a ribosome with the initiator tRNA in the P site, ready for elongation.

• Bacterial Translation Initiation:

  • Relatively simple process.

  • Initiation codon (AUG, sometimes GUG) is typically preceded by a Shine-Dalgarno sequence (ribosome-binding site) in the mRNA's 5' UTR.

  • The Shine-Dalgarno sequence base-pairs with the anti-Shine-Dalgarno sequence at the 3' end of the 16S rRNA in the small subunit, positioning the AUG in the P site.

  • Efficiency of translation is influenced by the complementarity of this interaction.

  • Initiator tRNA (f-Met-tRNA™") binding is guided by a small set of initiation factors (IF1, IF2, IF3).

• Eukaryotic Translation Initiation:

  • More complex than in bacteria.

  • Ribosomes identify the AUG start site using a scanning mechanism that begins at the 5' cap structure and depends on distinct factors (eIFs).

  • Eukaryotic 40S subunits do not directly bind upstream of the AUG using a Shine-Dalgarno-like sequence; the anti-Shine-Dalgarno tract is missing in 18S rRNA.

  • Internal ribosome entry sites (IRESs) allow direct ribosome binding near the AUG in some cases (e.g., viral mRNAs, stress conditions).

  • Requires many initiation factors (eIF1, 1A, 2, 2B, 3, 4A, 4B, 4E, 4G, 5, 5B), totaling at least 28 polypeptides in mammals.

  • Eukaryotic mRNAs have a 7-methyl guanosine cap at the 5' end and a poly(A) tail at the 3' end, both cotranscriptionally added.

  • These features, along with eIF4 family factors (eIF4E, PABP, eIF4A/4B, eIF4G), lead to the formation of a closed loop initiation complex on the mRNA, enhancing translation efficiency and serving as a quality control feature.

  • The 40S preinitiation complex (with initiator Met-tRNAi, eIFs, GTP-bound eIF2) binds the mRNA near the 5' cap via eIF3-eIF4G interaction and scans along the 5' UTR.

  • Recognition of an appropriate AUG (influenced by sequence context) triggers GTP hydrolysis on eIF2 (mediated by eIF5), a fidelity checkpoint.

  • Initiation factors dissociate, and the 60S subunit joins (promoted by eIF5B-GTP hydrolysis) to form the 80S initiation complex.

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

  • (Covered within "The Translation Cycle: the Ribosome in Action")

• Translation Termination, Recycling and Reinitiation:

  • (Covered within "The Translation Cycle: the Ribosome in Action")

Chapter 12: Translation Regulation

• Global Regulation of Initiation in Bacteria and Eukaryotes:

  • Translation is globally regulated in response to external conditions, particularly amino acid starvation.

  • Uncharged tRNA (lacking an attached amino acid) is an indicator of low amino acid levels and triggers a global shutdown of protein synthesis in both bacteria and eukaryotes, though via different mechanisms.

  • Bacteria: Uncharged tRNA binding to the ribosomal A site recruits RelA, which synthesizes (p)ppGpp ('magic spot'), leading to the stringent response: decreased stable RNA synthesis and induction of stress response genes (e.g., amino acid biosynthesis).

  • Eukaryotes: Uncharged tRNA binds to Gcn2 kinase, activating it to phosphorylate eIF2. Phosphorylated eIF2 binds more tightly to its GEF, eIF2B, effectively depleting active eIF2 and globally inhibiting translation initiation. This also paradoxically upregulates translation of certain mRNAs like GCN4.

  • Much global translational control in eukaryotes is achieved through modulation of eIF2 and its GEF partner eIF2B.

• Gene-specific translational regulation depends on sequence features in the mRNA:

  • The rate of synthesis of individual proteins is regulated according to cellular needs.

  • Rapid changes in protein synthesis can be achieved by regulating translation of existing mRNAs.

  • mRNA structural conformations can be critical for regulation.

  • In bacteria, regulation often involves obstructing the Shine-Dalgarno sequence in the 5' UTR.

  • Eukaryotic translation initiation involves both 5' and 3' UTRs, allowing for more diverse control mechanisms.

• Shine—Dalgarno sequestration is a common mechanism for the regulation of translation in bacteria:

  • If the Shine-Dalgarno sequence is inaccessible to the ribosome, initiation cannot occur.

  • Bacteria use various strategies to sequester the Shine-Dalgarno sequence:

    • mRNA 5' leader sequence structure: Temperature-dependent structural changes can expose or hide the Shine-Dalgarno sequence (e.g., prfA mRNA in Listeria monocytogenes).

    • Riboswitches: Small molecule metabolites (e.g., thiamine pyrophosphate binding to thiM/thiC mRNAs) bind to the 5' UTR, causing structural changes that sequester the Shine-Dalgarno sequence.

    • Small regulatory RNAs: Base-pairing with the 5' UTR can either sequester or reveal the Shine-Dalgarno sequence, often aided by RNA-binding proteins like Hfg. Base-pairing near the start codons can also inhibit initiation.

    • RNA-binding proteins: Proteins can bind to the mRNA and sterically block the Shine-Dalgarno sequence (e.g., ThrRS autoregulation of thrS mRNA translation).

• Initiation in eukaryotes can be regulated by blocking access to the ribosome-binding site:

  • Eukaryotes lack a direct equivalent to the bacterial Shine-Dalgarno ribosome-binding site; initiation typically occurs at the first AUG encountered during scanning from the 5' cap.

  • However, eukaryotic translation initiation can be hindered by blocking ribosome access to the 5' UTR initiation region.

  • Iron regulation of ferritin mRNA translation: Iron-regulatory proteins (IRPs) bind to iron response elements (IREs) in the 5' UTR when iron is scarce, preventing ribosome access to the AUG start site and thus inhibiting translation. When iron is plentiful, IRPs don't bind IREs, allowing translation.

• Upstream ORFs regulate the synthesis of Gcn4 protein in yeast:

  • The Gcn4 protein, a transcriptional activator of amino acid biosynthesis, is translationally controlled by multiple upstream ORFs (uORFs) in its 5' UTR.

  • Under non-starvation conditions (abundant eIF2-GTP-Met-tRNAi), ribosomes translate uORF1 and then reinitiate at uORFs 2-4, but the stop codons of uORFs 2-4 favor ribosome recycling, preventing translation of the GCN4 ORF.

  • Under amino acid starvation (low eIF2-GTP-Met-tRNAi), ribosomes may scan past uORFs 2-4 without reinitiating and then reinitiate at the GCN4 ORF, leading to increased Gcn4 protein synthesis.

• 3’ UTRs often contain sequences that regulate translation:

  • While 5' UTRs are more commonly involved in regulation in bacteria, the often longer 3' UTRs of eukaryotic mRNAs are crucial for regulating many developmentally important genes.

  • 3' UTR elements can recruit factors that disrupt the formation of the closed loop mRNA initiation complex, downregulating translation.

• Polyadenylation levels can be regulated by 3’ UTR binding proteins:

  • Example: Cytoplasmic polyadenylation element (CPE) in the 3' UTR of dormant mRNAs in Xenopus oocytes regulates polyadenylation and translation. CPEB protein binds CPE and interacts with Maskin, which inhibits eIF4E. Phosphorylation of CPEB recruits CPSF, leading to poly(A) tail extension. PABP then binds the poly(A) tail, recruits eIF4G, displaces Maskin, and allows translation initiation.

• RNAs or proteins bound to the 3’ UTR can regulate translation at multiple steps following polyadenylation:

  • 3' UTR regulatory sequences recruit various proteins that affect different steps of translation initiation or even elongation.

  • Example: DICE in the 3' UTR of lox mRNA binds hnRNP K and E1/E2, preventing large subunit joining.

  • Example: TCE in the 3' UTR of Nanos mRNA binds Smaug, which interacts with Cup (an eIF4E-binding protein), repressing translation by competing with eIF4G.

  • MicroRNAs (miRNAs) in eukaryotes bind to partially complementary sequences in the 3' UTRs of target mRNAs, repressing translation initiation and eventually leading to mRNA degradation.

• Viruses have evolved various ways to get their mRNAs translated by host ribosomes:

  • Viruses may inhibit host mRNA translation (e.g., picornaviruses cleaving eIF4G and PABP) and have evolved mechanisms for cap-independent translation of their own mRNAs.

  • Internal ribosome entry sites (IRESs) in the 5' UTR of viral mRNAs can directly recruit small ribosomal subunits, bypassing the need for some initiation factors and cap-dependent scanning. They bind where the Shine-Dalgarno interaction would occur in bacteria, positioning the start codon in the P site.

  • Some viruses (e.g., rotaviruses) produce mRNAs that compete for limiting translation factors.

  • Hepatitis C virus (HCV) and cricket paralysis virus (CrPV) initiate translation with a minimal set of initiation factors, with CrPV even independent of the eIF2 ternary complex.

• eIF2 dependent Met-tRNAi" loading is also a critical control point:

  • The availability of eIF2 for loading initiator Met-tRNAi into the ribosome is a key regulatory point.

  • Viral double-stranded RNA can activate PKR, which phosphorylates eIF2alpha, slowing down global protein synthesis.

  • Some viruses have evolved RNA elements in their 5' or 3' UTRs that mimic tRNA, allowing initiation at non-standard start codons independently of eIF2-mediated initiator tRNA loading (e.g., TYMV).