Protein Metabolism CH353 April 3, 2008

Genetic Code Codons of genetic code are in 5′→3′ direction on mRNA Initiation codon (AUG) is green 3 termination codons are red (UAA, UAG, UGA) 2 amino acids have unique codons (Trp - UGG, Met - AUG) Most codons are degenerate – more than one codon for the same amino acid 9 amino acids have 2 codons; 1 has 3 codons; 5 have 4 codons; 3 have 6 codons

Codon – Anticodon Recognition Wobble allows one tRNA (anticodon) to recognize multiple codons Base selection at 1st position of anticodon allows base-pairing with multiple bases in 3rd position of codon Wobble permits recognition of all 61 codons with only 32 tRNAs (including unique initiator tRNA)

The Ribosome A ribozyme for synthesizing proteins 50S and 30S subunits, with mRNA, with/without tRNAs bound at the E, P and A sites

Composition and Mass of Ribosomes Ribosomal subunits are named by their sedimentation velocities (S) rRNA forms the core of ribosomal subunits; proteins are secondary surface elements rRNA catalyzes peptide synthesis; no protein within 18 Å of active site 16S rRNA, tRNAs, mRNA cooperate for correct placement of amino acids Eukaryotic Ribosomes are ~56% larger than prokaryotic ribosomes Eukaryotic ribosomes have more proteins, larger rRNAs and an extra rRNA in 60S subunit

tRNA Secondary and Tertiary Structure Cloverleaf pattern of Watson-Crick base-pairing in tRNA, highlighting typical or invariant nucleotides 3D stucture of tRNA has ‘L’ shape; stacking anticodon arm + D arm, and TyC arm + amino acid arm

Major Stages of Protein Synthesis Activation of Amino Acids activate carboxyl group of amino acid for peptide synthesis establish link between amino acid and biological information Initiation of Translation join initiation codon of mRNA to ribosome and initator tRNA Elongation of Translation recruit aminoacyl-tRNA based on codon of mRNA template form peptide bond and advance to next codon Termination of Translation recruit termination factors to stop codon release completed polypeptide Folding and Post-translational Processing fold into active form; proteolytic processing; modify amino acids

Stage 1: Aminoacylation of tRNA Overall Reaction: ∆G′º = -29 kJ/mol Amino acid + tRNA + ATP → aminoacyl-tRNA + AMP + PPi PPi + H2O → 2 Pi Aminoacyl-tRNA Synthetases catalyze at least 2 reactions: amino acids activation with ATP, and aminoacyl transfer to tRNA 2 classes of aminoacyl-tRNA synthetases: Class I: Arg, Cys, Gln, Glu, Ile, Leu, Met, Trp, Tyr, Val Class II: Ala, Gly, Asn, Asp, His, Lys, Phe, Pro, Ser, Thr

Aminoacylation of tRNA Class I aminoacyl-tRNA synthetases Initial esterification on 2′ OH Class II aminoacyl-tRNA synthetases Initial esterification on 3′ OH

Proofreading by Aminoacyl-tRNA Synthases Correct connection between amino acid and anticodon needs to be made here (no amino acid proofreading on ribosome) Interaction with amino acid at 2 levels of specificity; binding to amino acid and to aminoacyl-AMP Provides multiplicative lower error rate of ~10-4 Interaction with tRNAs: multiple tRNAs but one synthetase per amino acid; specificity determined by characteristic nucleotides and structure of tRNAs

Stage 2: Translation Initiation Step 1: 30S subunit binds to IF-1 and IF-3 and then to mRNA (AUG at P site) IF-1 blocks A site, IF-3 prevents premature association with 50S subunit Step 2: IF-2–GTP binds to fMet-tRNAfMet, which facilitates binding to 30S complex tRNA anticodon pairs with codon of mRNA Step 3: GTP is hydrolyzed; IF-1, -2, -3 dissociate 30S complex combines with 50S subunit to form 70S initiation complex

Translation Initiation on Bacterial mRNA Correct positioning of 30S ribosomal subunit at initiation codon requires an upstream Shine-Dalgarno sequence, complementary to 3′ end of 16S rRNA Extent of complementarity and spacing relative to initiation codon determine initiation efficiency; requirements are species specific (more is not always better) AUG is not always initiation codon (GUG and UUG can be used), but fMet-tRNAfMet is used regardless of codon 3’ A U U C C U C C A . . . | | | | | | | | | 3’ End of 16S rRNA

Translation Initiation on Eukaryotic mRNA Initiation codon is nearly always the 5′-terminal AUG Kozak sequence facilitates initiation (5′) ACCAUGG (3′) 5′-terminal cap and 3′-terminal poly(A) are essential eIF4F complex (4A, 4B, 4E, 4G) binds to cap and to eIF3 Scanning for AUG requires ATP and eIF4A (helicase) eIF2 similar role as IF-2, binding Met-tRNAiMet (GTP required) eIF5 involved in assembly of 80S ribosome (GTP required)

Stage 3: Translation Elongation Step 1: Binding of Aminoacyl-tRNA aminoacyl-tRNA binds to EF-Tu–GTP resulting assembly binds to A site of 70S initiation complex GTP is hydrolyzed; EF-Tu is released; EF-Tu–GTP regenerated with GTP and EF-Ts Proofreading of codon-anticodon interaction: aa-tRNA complexes with EF-Tu-GTP and with EF-Tu-GDP exist for few milliseconds If correct codon-anticodon is not found during this time, complex dissociates 18S rRNA involved in correct base pairing eEF1a and eEF1bg are eukaryotic analogs

Stage 3: Translation Elongation Step 2: Peptide Bond Formation Transfer of the acyl peptide at the P site to the aminoacyl-tRNA at the A site Nucleophilic attack by amino group (A site) on acyl group (P site) with tRNA as leaving group (remaining bound to P site) Reaction catalyzed by 23S (or 28S) rRNA Step 3: Translocation Ribosome shifts one codon toward the 3′ end of the mRNA (peptidyl-tRNA to P site and empty tRNA to E site) EF-G–GTP binding to A site mimics the EF-Tu-tRNA complex displacing peptidyl-tRNA eEF2 is eukaryotic factor analogous to EF-G

Stage 4: Translation Termination 3 Termination factors (release factors) proteins RF-1, RF-2 and RF-3 facilitate: hydrolysis of terminal peptidyl-tRNA bond release of polypeptide and tRNA dissociation of 70S ribosome into subunits RF-1 binds UAG or UAA; RF-2 binds UGA or UAA RF-1 or RF-2 binds to stop codon, causing peptidyl transferase ribozyme to use water instead of aminoacyl-tRNA as nucleophile (mimic tRNA structure – like EF-G) RF-3 may function in ribosome dissociation eRF performs all functions in eukaryotes

Polysomes (Poly-ribosomes) Multiple ribosomes simultaneously translating a single mRNA provides: Efficient protein synthesis 10 to 100 ribosomes producing protein from each mRNA Stability of mRNA Close spacing of ribosomes protect mRNA from ribonucleases Polysomes occur in both prokaryotic and eukaryotic translation

Coupled Transcription and Translation Eukaryotic transcription occurs in nucleus but translation occurs in cytosol – requires translocation of processed mRNA to cytosol Prokaryotes have no nuclei so the nascent mRNA can be translated while it is being transcribed Eukaryotic mRNA nearly always encode 1 protein (moncistronic) Prokaryotes typically have multiple coding regions in a single mRNA Operons transcribe multiple coding regions into one mRNA Operons often coordinate the biosynthesis of proteins serving one function, e.g. multisubunit proteins or metabolic pathways

Stage 5: Post-Translational Processing Cytoplasmic Modifications Proteolytic processing hydrolysis of N- and C-terminal amino acids; deformylating fMet Amino acid modification phosphorylation, methylation, acetylation Attachment of lipids isoprenylation, N-myristoylation, C-palmitoylation Attachment of prosthetic groups activation of enzymes and proteins (e.g. coenzymes, heme)

Stage 5: Post-Translational Processing Secretion and Targeting Signal (or target) peptide removal hydrolysis of signal or target peptide during secretion Attachment of carbohydrate groups and lipids O-linked (Ser or Thr) and N-linked (Asn) glycosylations C-terminal GPI (glycosyl phosphodylinositol) anchors Disulfide bond formation Disulfide-bonded proteins are usually lumenal or extracellular Proteolytic Processing activation of pro-enzymes and pro-hormones Amino acid modification Glu g-carboxylation, Pro hydroxylation, others

Processes Affecting Protein Levels Synthesis of primary RNA transcript (transcription) Posttranscriptional modification of mRNA Degradation of mRNA Protein synthesis (translation) Posttranslational modification of proteins Protein targeting and transport Protein degradation