Translation

Hybrid States

-intermediate stage: tRNAs occupying partially translocated hybrid state -anticodon ends still reside in A and P sites of small subunit, acceptor ends have moved into P and E sites of large subunit -tRNAs occupy A/P and P/E hybrid sites -shift from nonratcheted hybrid state to hybrid, ratcheted state occurs spontaneously, without involvement of other factors -->ribosomes can spontaneously oscillate back and forth -once it has shifted to hybrid state, GTP-bound elongation factor (EF-G/eEF2) binds to ribosome, stabilizing ribosome in ratcheted state, preventing movement of tRNAs back to classic A/A and P/P conformation

Protein Folding

-linear sequence of amino acids contains all info required for formation of polypeptide's 3D conformation -events progress toward states of lower energy: tertiary structure assumed after folding is accessible structure with lowest energy, most thermodynamically stable

mRNA Surveillance

-nonsense mutations: termination codons formed by single base changes -->premature termination codons -mRNA containing mutations translated once before destroyed by nonsense-mediated decay

Prokaryotic Initiation: 3. Assembling the Complete Initiation Complex

-once initiator tRNA bound to AUG and IF3 displaced, large subunit joins complex, GTP bound to IF2 hydrolyzed -GTP hydrolysis drives conformational changes in ribosome required for release of IF2-GDP, allowing translation to proceed

Dynamics of Protein Folding

-proteins initially explore wide range of conformations when they first begin to fold, eventually funnel into restricted set of possible configurations 1. protein folding initiated by interactions among neighbouring residues that lead to formation of secondary structure of molecule 2. once alpha helices and beta sheets form, subsequent folding driven by hydrophobic interactions that bury nonpolar residues together in central core of protein 3. hydrophobic collapse of polypeptide forms compact structure, backbone adopts native-like topology -->significant secondary structure develops

Bacterial Initiation Factors

-require 3 initiation factors that bind to large subunit 1. IFI: stabilizes attachment of small subunit to mRNA, prevents initiator aa-tRNA from entering wrong site on ribosome 2. IF2: GTP-binding protein required for attachment of 1st aminoacyl-tRNA 3. IF3: prevents large subunit from joining prematurely to small subunit, facilitates entry of appropriate initiator aa-tRNA

The Assembly of Ribosomal Subunits

-ribosomes made of RNA and protein -all ribosomes composed of 2 subunits of different size, highly irregular shape -->large (50S): 2 molecules of RNA, 32 different proteins) -->small (30S): 1 molecule of RNA, 21 different proteins -small subunit contains all info necessary for assembly of entire particle

Molecular Chaperones

-several families of proteins whose function is to help unfolded/misfolded proteins achieve proper 3D conformation -selectively bind to short stretches of hydrophobic amino acids exposed in non-native proteins but buried in proteins with native conformation

tRNA Charging

-tRNA charging: each tRNA molecule must be attached to cognate amino acid -amino acids covalently linked to 3' ends of cognate tRNAs by aminoacyl-tRNA synthetase -->20, 1 for each amino acid -->each capable of charging all tRNAs appropriate for that amino acid -recognition achieved by: -->identifying tRNA anticodon nucleotides -->recognizing nucleotide sequence of acceptor stem/arm -->reading nucleotide sequences at additional positions on tRNA

Antibiotics

-target for most antibiotics is translation in prokaryotic ribosome -many modes of resistance: -->altering target (drug has target on prokaryotic ribosomes, mutations, selection over time) -->pumping antibiotic out of cell before it destroys all ribosomal activity

Release Factors

-termination requires release factors: class I RFs (recognize codons in A site), class II RFs (GTP-binding proteins) -bacteria have 2 class I RFs: RF1 (recognizes UAA and UAG), RF2 (recognizes UAA and UGA) -eukaryotes have single class I RF (eRFI), recognizes all stop codons

Role of tRNAs

-translation requires info encoded in nucleotide sequence of mRNA decoded and used to direct sequential assembly of amino acids into polypeptide chain -decoding info in mRNA accomplished by tRNAs, as adaptors -each tRNA linked to specific amino acid (aa-tRNA), same tRNA able to recognize particular codon in mRNA -interaction between successive codons in mRNA and specific aa-tRNAs leads to synthesis of polypeptide with ordered sequence of amino acids

Termination of Translation in Humans

-we have release factor which resembles tRNA, but is made out of protein instead of ribonucelotides -example of molecular mimicry -->because it looks similar, can enter A site, works like prokaryote -->polypeptide is released, small subunit can't ratchet, ribosome falls apart

How mRNA Distinguishes PTC

-when intron removed by spliceosome, complex of proteins deposited on transcript upstream from newly formed exon-exon junction, exon-junction complex (EJC) stays with mRNA until translated -normal mNRA: termination codon present in last exon, last EJC upstream from that site -->as mRNA undergoes initial round of translation, EJCs inactivated by advancing ribosome; any message with EJC left marked for degradation -->normal stop codons occur after last intron, downstream of last EJC, all EJCs removed -PTC: ribosome stops at site of mutation and dissociates, leaving any EJCs attached to mRNA downstream of site of premature termination

Polyribosomes

-when mRNA in process of being translated, number of ribosomes attached along length of mRNA thread (polyribosome/polysome) -each ribosome initially assembled from subunits at initiation codon, moves toward 3' end of mRNA until it reaches termination codon -->another ribosome attaches to mRNA, begins translation activity -3D arrangement and orientation of ribosomes within polysome highly ordered -->densely packed, double-row array -->each individual ribosome oriented so nascent polypeptide situated at outer surface facing cytosol (maximizes distance between nascent chains, minimizing likelihood that nascent chains will aggregate)

Termination

1. class I RFs enter A site, conserved tripeptide at one end of RF interacts with stop codon in A site, triggers conformational change affecting several nucleotides of mRNA of small subunit 2. ester bond linking nascent polypeptide chain to tRNA is hydrolyzed, completed polypeptide released 3. hydrolysis of GTP bound to class II RF (RF3 or eRF3) leads to release of class I RF from A site 4. release of deacylated tRNA from P site, dissociation of mRNA from ribosome, disassembly of ribosome into subunits in preparation for another round of translation

Eukaryotic Initiation Factors

1. eIF4E: binds to 5' ca[ 2. eIF4A: moves along 5' end of message, removing double-stranded regions that would interfere with movement of 43S complex along mRNA -eIF4G: linker between 5' and 3' ends of mRNA (converts linear mRNA into circular)

Properties of tRNA

1. same length, continuous RNA sequence (one 5' end, one 3' end) 2. significant percentage of unusual bases resulting from enzymatic modifications of 1 of 4 standard bases after incorporation into RNA chain -->concentrated in loops, disrupt H bond formation 3. have sequences of nucleotides in 1 part of molecule complementary to sequences located in other parts 4. become folded to form 2D cloverleaf structure 5. single-stranded, double-stranded in certain parts -->important single-stranded areas: 3' end (amino acid attached), anticodon area

Prokaryotic Initiation: 1. Bringing the Small Ribosomal Subunit to the Initiation Codon

-1st step: binding of small subunit to 1st AUG sequence (initiation codon) -bacterial mRNAs possess Shine-Dalgarno sequence, resides 5-10 nucleotides before initiation codon, complementary to sequence of nucleotides near 3' end of rRNA of small subunit -interactions between complementary sequences on mRNA and rRNA positions large subunit at initiation codon

Stop Codons

-3 codons function as stop codons, terminate polypeptide assembly -->no tRNAs whose anticodons complementary to stop codons -when ribosome reaches UAA, UAG, UGA, signal read to stop further elongation, release polypeptide associated with last tRNA

Prokaryotic Initiation: 2. Bringing the First aa-tRNA into the Ribosome

-AUG only codon for methionine: 1st amino acid incorporated, removed from majority of proteins -2 methionyl-tRNAs: 1 to initiate protein synthesis, 1 to incorporate methionyl residues into interior of polypeptide -initiator aa-tRNA positioned by IF2 within P site, anticodon loop of tRNA binds to AUG codon of mRNA, IF1 and IF3 released

Protein Synthesis in Prokaryotes

-activities tightly coupled -protein synthesis begins on mRNA templates before mRNA completely synthesized -synthesis of mRNA proceeds in same direction as movement of ribosomes translating message (5' --> 3') -as soon as RNA molecule has begun to be produced, 5' end available for attachment of ribosomes

Structure of tRNA

-all mature tRNAs have sequence CCA at 3' end -fold into unique and defined tertiary structure -->constructed of 2 double helices arranged in L-shape -each has unique features, amino acid can become attached to cognate tRNA -translate sequence of mRNA codons into sequence of amino acid residues

Chaperones in Polypeptide Chains

-chaperones of Hsp70 family bind to elongating polypeptide chains as they emerge from exit channel within large subunit -prevent partially formed polypeptides from binding to other proteins in cytosol (cause them to aggregate or misfold) -once synthesis complete, many proteins released by chaperones into cytosol, spontaneously fold into native state -other proteins repeatedly bound and released by chaperones until they reach fully folded state -many larger polypeptides transferred to chaperonin -->cylindrical protein complexes, contain chambers in which newly synthesized polypeptides can fold without interference from other macromolecules

Elongation: 4. Releasing the Deacylated tRNA

-deacylated tRNA leaves ribosome, emptying E site -for each cycle of elongation, at least 2 molecules GTP hydrolyzed: 1 during aminoacyl-tRNA selection, 1 during translocation 1. once peptidyl-tRNA has moved to P site by translocation, A site open to entry of another aminoacyl-tRNA 2. once 3rd charged tRNA associated with mRNA in A site, dipeptide from tRNA of P site displaced by aa-tRNA of A site, forming 2nd peptide bond and tripeptide attached to tRNA of A site -->tRNA in P site devoid of amino acid 3. peptide bond formation followed by translocation of ribosome to 4th codon, release of deacylated tRNA, cycle ready to begin again

Elongation

-each tRNA steps through 3 positions on ribosome: binds at A site first; after peptide bond formation shifts to P site; after next round of peptide bond formation moves to E site; exits ribosome

Elongation: 2. Peptide Bond Formation

-end 1st step: 2 amino acids, attached to separate tRNAs, aligned to chemically interact -for 1st peptide bond, P site holds initiator tRNA; for all other peptide bonds, P site holds tRNA attached to growing peptide chain -A site holds newly arriving aminoacyl-tRNA that needs to be added for chain to elongate 1. formation of peptide bond between 2 amino acids: amine N of aa-tRNA in A site carries out nucleophilic attack on carbonyl C of aa-tRNA in P site, displacing P-site tRNA 2. tRNA bound in A site has attached dipeptide, tRNA in P site deacylated -reaction catalyzed by peptidyl transferase (component of large subunit) -->performed by P site tRNA with assistance from nucleotides of larger rRNA molecule

Elongation: 3. Translocation

-formation of 1st peptide bond leaves 1 end of tRNA molecule of A site attached to complementary codon on mRNA, other end attached to dipeptide, tRNA of P site devoid of amino acid -ratchet-like motion of small subunit relative to large: ribosome moves 1 codon along mRNA in 5' --> 3' direction -accompanied by movement of dipeptidyl-tRNA from A to P site of ribosome and deacylated tRNA from P to E site -hydrolysis of bound GTP generates conformational change that moves mRNA and associated anticodon loops of tRNAs relative to small subunit, places bound tRNAs in E/E and P/P states, leaving A site empty -->ribosome reset to nonratcheted state -->EF-G-GDP dissociates

The Role of the Ribosome

-highly irregular structure -each ribosome has 3 sites for association with tRNA molecules: A (aminoacyl), P (peptidyl), E (exit) -tRNAs bind within these sites, span gap between 2 ribosomal subunits -other major features: 1. interface between subunits cavity that is lined by RNA 2. active site: amino acids covalently linked, consists of RNA 3. mRNA situated in narrow groove that winds around neck of small subunit, passing through A, P, and E sites -->prior to entering A site, mRNA stripped of secondary structure by helicase 4. tunnel runs through core of large subunit beginning at active site, provides passage for translocation of elongating polypeptide through ribosomes 5. most proteins of ribosomal subunits have multiple RNA-binding sites, stabilize tertiary structure of rRNAs

Role of Elongation Factors

-improve speed and efficiency (EF-G prokaryotes/EF2 eukaryotes) -error checking function (EF-Tu prokaryotes/EF1A eukaryotes)

Wobble Hypothesis

-interchangability of base of 3rd position: same tRNA recognizes more than 1 codon -wobble hypothesis: steric requirement between anticodon of tRNA and codon of mRNA strict for first 2 positions, more flexible at 3rd position -2 codons that specify same amino acid and differ only at 3rd position use same tRNA

Initiation in Eukaryotes

1. several eIFs bind to small subunit, prepare for binding to mRNA 2. initiator tRNA linked to methionine binds to subunit prior to interaction with mRNA -->enters P site of subunit in association with eIF2-GTP -->small subunit with associated initiation factors and charged tRNA (form 43S PIC0 ready to find 5' end of mRNA (with cap) 3. 43S complex recruited to mRNA with help of initiation factors bound to mRNA 4. once 43S complex binds to 5' end, complex scans message until it reaches sequence with AUG 5. GTP bound to eIF2 is hydrolyzed, large subunit joins complex -->formation of ribosome requires activity of another GTP-binding protein (eIF5B-GTP), bound GTP also hydrolyzed 6. hydrolysis of 2 bound GTPs and formation of complete ribosome accompanied by release of all initiation factors -->leave anticodon and initiator tRNA bound to AUG in P site, complex ready for elongation

Elongation: 1. Aminoacyl-tRNA Selection

1. with charged initiator tRNA in P site, ribosome available for entry of 2nd aminoacyl-tRNA into vacant A site -requires GTPase (EF-Tu/eEF1A) -->delivers aminoacyl-tRNAs to A site -->any aminoacyl-tRNA-Tu-GTP complex can enter site; only those whose anticodon is complementary to mRNA codon in A site will trigger conformational changes within ribosome that cause tRNA to remain bound to mRNA in decoding centre (H bonding must be correct) 2. once proper aminoacyl-tRNA-Tu-GTP bound to mRNA codon, GTP hydrolyzed, Tu-GDP complex released, leaving newly arrived aa-tRNA in A site