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Chapter 30

Notes Chapter 30 (Lectures 17-21) - Biochem 2B03.docx

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Margaret Fahnestock

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Biochem 2B03 2012 8Part 4: Information Transfer Chapter 30: Protein Synthesis Refer to Chapter 29 Notes 29.3-29.4 RPB1 and its C-Terminal Domain (CTD)  CTD of RPB1 is essential to the function of RNAP II  YSPTSPS repeated 60 times (human)  RNAP II only initiates transcription when the CTD is not phosphorylated  Elongation proceeds only after phosphorylation of CTD, suggesting phosphorylation triggers initiation to elongation conversion  Following termination, a phosphatase recycles RNAP II for initiation CTD also play a prominent role in orchestrating subsequent events for processing the transcript (Capping, splicing, Polyadenylation)  General structure of amino acids o Cα is a chiral center (with one exception) The 20 Common Amino Acids  Non-polar amino acids (hydrophobic) 1 Biochem 2B03 2012  Polar, uncharged amino acids  Acidic amino acids  Basic amino acids Peptide Bond Formation  How is the nucleotide sequence of an mRNA molecule translated into the amino acid sequence of a protein molecule? 30.1 What is the Genetic Code? 2 Biochem 2B03 2012 What is the nature of the code?  Genetic experiments demonstrated a triple code  RNA has four bases C, G, A, U  4 ways of combining these in pairs 3  4 ways of combining these in triplets  In 1960 you couldn’t sequence DNA but could sequence protein  Three bases codes for one amino acid  Code is not overlapping  Base sequence is read from a fixed starting point without punctuation  Code is degenerate (each amino acid can be coded for by several triplets) In vitro transition reactions  Prime them with synthetic RNAs of defined sequence RNA Sequence Protein Produced oUUUUUUUUU…. Phe Phe Phe Phe… AAAAAAAAA… Lys Lys Lys Lys… GGGGGGGGG… Gly Gly Gly Gly… CCCCCCCCCC… Pro Pro Pro Pro… UAUAUAUAU… Tyr Ile Tyr Ile… UAUUUUUAUUUU Tyr Phe Tyr Phe UUU Phe AAA Lys GGG Gly CCC Pro UAU Tyr The Genetic Code  Note: termination codons, start codon, redundancy: Trp has the only unique codon  Codons – triplets of bases in mRNA; read 5’3’  All codons have meaning; of 64 codons, 61 code for particular amino acids, 3 are nonsense codons serving as termination codons (stop signal)  Genetic code is o Unambiguous – each codon only codes for one amino acid o Degenerate – each amino acid (exception to Trp and Met) is coded for by more than one codon; synonymous codons  Codons representing the same amino acid or chemically similar amino acids tend to be similar in sequence  Eg/ GGx codons – Gly; UCx codons – Ser  Codons with pyrimidine as 2base – likely contain hydrophobic side chain  Codons with purine as 2base – likely contain polar or charged amino acids  Mutations due to single base changes are less harmful 3 Biochem 2B03 2012 o Universal – codon assignments are same throughout all organisms  Evidence that all organisms evolved from a common primordial ancestors How To Translate an mRNA – Part I  Sequence is composed of triples o UAU CAC AUG CAC GUA CUG UGA… o Tyr His Met His Val Leu stop + - NH3 COO end How To Translate a mRNA – Part II  Recognizing the start site of the coding sequence:  “Alignment” of the 5’ ends of transcripts reveals conserved sequences o Purine-rich sequence near 5’ end of transcript – “Shine Delgarno” site o 4-8 nt upstream of an AUG codon for Methionine The Genetic Code is generally regarded as universal  Minor exceptions found in mitochondria: o UGA codes for Trp rather than termination (animal mitos) o AUA codes for met, rather than Ile (fungal mitos) o AGA codes for termination rather than arg (animal mitos) o AGA codes for ser rather than arg (fruit flies mitos)  Variation in the GC richness causes bias in codon usage o Important biotechnological implications B. subtilis – 60% AT S. coelicolor – 73% GC GC  E. coli and humans nonrandom usage of codons  Occurrence of codons in E. coli mRNAs correlates well with the relative abundance of the tRNAs that read them tRNAs – the bridge between the codons and the amino acids 4 Biochem 2B03 2012  D-loop – contains dihydrouridine variable length 7-12 nt  TyC-loop – contains pseudouracil (Ψ) contains thymine  Variable loop – present in some tRNAs  Anticodon loop – recognizes codon  Acceptor stem – attaches to amino acid CCA 3’  Note – non-W/C base-pairs  Note: R = purine, Y=pyrimidine Structural Complexity in RNA  Unusual bases found in tRNAs (and rRNA)  Stems, loops, bulges and junctions are the four basic secondary structural elements in RNA  Structural motifs: U-turns, tetraloops and bulges  Regions where several stemloop structures meet: junctions  Other tertiary structural motifs arise from coaxial stacking pseudoknot formation and ribose zippers  tRNA is like a “globular RNA” – it has 2° and 3° structure like proteins 30.2 How Is an Amino Acid Matched With It’s Proper tRNA tRNAs and aminoacyl tRNA synthetases  Aminoacyl tRNA o Bridge the genes and proteins o Recognize the codons (via anticodon loop) o Position amino acids in ribosome for peptide bond formation  Amino acid covalently attaches to the 3’ end – “acceptor step”  Attachment is mediated by amino acyl tRNA synthetase enzymes o Aminoacyl-tRNA synthetases catalyze ATP-dependent attachment of specific amino acids to 3’ end of cognate tRNA molecules to form aminoacyl-tRNA, AMP and PPi(which becomes 2 Pi) 5 Biochem 2B03 2012  Amino acylation of tRNA is a two-step reaction i. Formation of the aminoacyl-adenylate – this is an “activated” form of the amino acid (peptide bond formed) o Note – production of PPi ii. Transfer of the activated amino acid to the 2’-OH or 3’-OH of the tRNA two mechanisms for this step (class I and class II) Aminoacyl-tRNA Synthetases  Aminoacyl-tRNA synthetases fall into classes on the basis of similar amino acid sequence motifs, oligomeric state and acylation function  Class I enzymes first add the amino acid to the 2’-OH of the terminal adenylate residue of tRNA before shifting it to the 3’OH o Bind to the tRNA acceptor stem helix from the minor-groove side  Class II enzymes add it directly to the 3’-OH o Bind to the tRNA acceptor stem from the major-groove side  The “class rule”: each amino acid is loaded onto tRNA by either class I or class II enzyme – there are exceptions to this rule  The class I and class I enzymes constitute conserved families  Note: the families have distinct means of interacting with cognate tRNAs  Exception to the “class rule” class I and II enzymes for lysine in some Archea 6 Biochem 2B03 2012 How are tRNAs matched with their cognate amino acids?  The code by which each aminoacyl-tRNA synthetase matches up its amino acid with tRNAs constitutes a secondary genetic code  Aminoacyl-tRNA synthetases interpret the second genetic code  Diverse, tRNA-specific mechanisms for recognition  For most tRNAs there are several recognition elements Specificity Determinants – how aminoacyl synthetases discriminate between tRNAs  Structural features that permit synthetases to recognize their cognate tRNAs are not universal  A set of tRNA sequence elements are recognized by its specific aminoacyl-tRNA synthetase o At least one base in the anticodon o One or more of the three base pairs in the acceptor stem o The base at canonical position 73 – discriminator base (unpaired base preceding the CCA/acceptor end)  These sequence elements can also act as negative determinants that prohibit binding and aminoacylation by other (noncognate) aminoacyl-tRNA synthetases  Filled circles = positions recognized by the AAS Position 73 Anticodon Loop Acceptor Stem Variable Loop  E. coli Glutaminyl-tRNA Synthetase tRNA complex o Note agreement between tRNA “code” and tRNA/synthetase structure o Class I enzyme o Glutaminyl-tRNA Glnsynthetase shares a continuous interaction with its cognate tRNA that extends from the anticodon to the acceptor stem along the entire inside of the L-shaped tRNA A Single G:U Base Pair Defines tRNAls  Especially simple case: tRNAlNon-WC base pair at position 70 is the only essential determinant of the tRNA/AA tRNA synthetase match  All ala tRNAs have this – mutations at this position abolish recognition 7 Biochem 2B03 2012 How does the aa-tRNA synthetase prevent mis-charging of tRNAs?  Amino acid structures are often quite similar Tyr and Phe  Charging of Phe by the tRNA Tyrsynthetase doesn’t occur because stable binding of the amino acid by the amino acid activating site (reaction #1) requires the OH group Val and Ile Ile  Val and Ile – in E. coli, [Val] = 5x [Ila] – at this ratio, tsynthetase binds and activates valine 3% of the time  But the frequency of mis-chanrging of Ile tRNA is <<0.1% o Solution: Proof-Reading Mechanism Ile  Immediately after the charging reaction the Val-tRNA enters the proof-reading site in the AA-tRNA-synthetase  Here, hydrolysis removes the incorrect amino acid from the tRNA Ile  The correctly charged Ile-tRNA does not fit in this site due to the extra methyl group on the side chain Val and Thr  These amino acids are very similar in size and shape but differ in side-chain hydrophobicity  Accuracy is accomplished by recognition of this property at two points Val 1. The acylation site (reaction 1) of the tRNAis hydrophobic – favours interaction with valine over threonine 2. The proof-reading site of the tRNAalis hydrophilic – favours interaction with threonine  The result of these mechanisms is that the charging of tRNAs is generally very accurate  This ensures that the proteins that are produced by the ribosome are an accurate reflection of the sequence of the genes  Since evolution selects genotypes on the basis of phenotype this is extremely important! 30.4 What Is The Structure of Ribosomes and How Are They Assembled? Transition is carried out on massive enzymes called ribosomes  Ribosomes – compact ribonucleoprotein particles; in cytosol of all cells, mitochondrial matrix and stroma of chloroplasts o Move along mRNA templates, orchestrating the interactions between successive codons and the corresponding anticodons presented by aminoacyl-tRNAs o Align successive amino acids via codon-anticodon recognition, ribosomes also catalyze the formation of peptide bonds between the growing peptide chain and incoming amino acids 8 Biochem 2B03 2012  E. coli ribosome: 25 nm diameter
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