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Computational Biology I LSM5191 Aylwin Ng, D.Phil Lecture Notes: Transcriptome: Molecular Biology of Gene Expression II TRANSLATION RIBOSOMES: protein synthesizing machines • Translation takes place on defined cytoplasmic organelles called RIBOSOMES. ROLES OF RNA IN TRANSLATION Three types of RNA molecules perform different but complementary roles in translation: Messenger RNA (mRNA) carries information copied from DNA. Transfer RNA (tRNA) deciphers the code and delivers the appropriate amino acid specified by a codon (a unit of 3 nucleotides). Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes, structures that function as protein-synthesizing machines What is a Codon? • A unit of 3 nucleotides. • Each Codon (except for stop codons) encodes an amino acid. TRANSFER RNA (tRNA) • Forms a vital link between mRNA & the growing polypeptide chain. • Anti-codon (on tRNA) base-pairs with CODON (on mRNA ). • Amino-acid (a.a.) is attached at 3’-end of tRNA. • Specificity: Each tRNA type is specific for only the a.a. it carries: E.g.: tRNAMet carries only methione; and tRNAGly only glycine. • 50 different tRNAs in eukaryotes. • But only 20 amino acids are designated by the genetic code. Codon degeneracy. Different tRNA types can carry the same amino-acid (due to ‘wobble’ basepairing). • e.g. tRNAGly1 and tRNAGly2 are both specific for glycine. • All mature tRNA ends with –CCA. (CCA added by tRNA nucleotidyl-transferase). TRANSFER RNA (tRNA) Cloverleaf structure (dihydrouridine) (ThymidinepseudoU-cytidine) 3-D Structure Aminoacylation (‘charging’) of tRNA • Attachment of amino-acid (a.a) to tRNA ensures specificity. • Catalysed by aminoacyl-tRNA synthetases. • Each tRNA is recognised by a specific aminoacyl-tRNA synthetase. • Aminoacylation occurs in 2 steps. Step 1: • Formation of activated a.a. intermediate; • a.a linked to enzyme via highenergy bond. Step 2: a.a transferred to 3’-end of tRNA. Overall rxn: enz a.a + ATP + tRNA Æ Aminoacyl-tRNA + AMP + 2Pi Step 1 Æ Step 2 Æ Codon & tRNA anticodon recognition • Specificity of aminoacylation Æ ensures tRNA carries the right a.a. denoted by the codon the tRNA pairs with. ‘Wobble’ base-pairing occurs ‘Wobble’ = non-standard (non-Watson-Crick) base pairing: The ‘Wobble’ rules: • G-U pairing acceptable. • Inosine (I), [a modified version of Guanosine], can pair with A, C and U. ‘Wobble’ base-pairing G-U base-pairing Alanine codons 5’- GCC GCU GCA GCG Enables the 4 codons for alanine to be decoded by just 2 tRNAs. Inosine base-pairs with A, C and U Enables the 3 codons for isoleucine to be decoded by just one tRNA. THE GENETIC CODE TRANSLATION INITIATION In Prokaryotes including bacteria, Translation is initiated when the small ribosome subunit + initiation factor (IF3) binds to Shine-Dalgarno seq. (5’-AGGAGGU-3’) This seq. is 3-10 nucleotides upstream of the initiation codon (start site). Initiator tRNA is ‘charged’ with N-formylmethionine or methionine. In Eukaryotes, Ribosome binds to the 5’ end of mRNA by recognizing the methylated cap. Ribosome moves along mRNA until it encounters AUG within Kozak seq (5’-ACCAUGG-3’) Æ initiation of translation. Initiator tRNA is ‘charged’ with methionine. TRANSLATION INITIATION (Eukaryotes) Cap 5’ (A)n 3’ AUG (A)n 3’ AUG eIF4E eIF4A eIF4F complex tRNA eIF4G eIF4E eIF2 40S (A)n 3’ AUG tRNA eIF4F complex eIF4A eIF2 40S eIF4G eIF4E (A)n 3’ AUG eIF2 60S AUG 40S (A)n 3’ Translation ELONGATION OF TRANSLATION • Mechanism very similar in bacteria and eukaryotes. Peptide bond formation catalysed by peptidyl transferase eEF-1 Animation clip EXERCISES Exercise 1a: 5’- GTAGCCTACCCATAGG -3’ If mRNA is transcribed from this DNA using the complementary strand as a template, what will be the seq. of the mRNA? 5’ – GUAGCCUACCCAUAGG - 3’ What peptide will be made if translation started exactly at the 5’ end of this mRNA? (assume no start codon is req.d). Valine(V) – Alanine(A) – Tyrosine(Y) – Proline(P) Exercise 1b: 5’ – GUAGCCUACCCAUAGG - 3’ Potentially, how many different peptides are encoded in this mRNA? 3 different peptides, since there are 3 different reading frames. 5’ – GUAGCCUACCCAUAGG - 3’ V A Y P * (Frame 1) * P T H R (Frame 2) S L P I (Frame 3) Six peptides … if the stretch of DNA (in Exercise 1a) is also transcribed in the opposite direction, i.e. both strands serving as templates for transcription. Exercise 2: If the anti-codon of a tRNA has this sequence: 5’- G C U –3’ Which could be the likely corresponding codon sequence on the mRNA? (1) 5’- C G A –3’ (2) 5’- A G C –3’ (3) 5’- C G T –3’ (2) and (4) (4) 5’- A G U –3’ Which amino acid is the tRNA likely to be specific for? Serine Locating genes by scanning Open Reading Frame (ORF) Human Interleukin-2 (IL-2) gene - promoter, exon 1 and partial cds (Accession Number: AF031845) 1 61 121 181 241 301 361 421 481 tatgacaaag aaaactgttt ctaatgtaac aaattccaaa gtctttgaaa attaacagta cacagtaacc aagtcttgca acaactggag aaaattttct catacagaag aaagagggat gagtcatcag atatgtgtaa taaattgcat tcaactcctg cttgtcacaa catttactgc gagttacttt gcgttaattg ttcacctaca aagaggaaaa tatgtaaaac ctcttgttca ccacaatgta acagtgcacc tgtatcccca catgaattag tccattcagt atgaaggtaa attttgacac agagttccct caggatgcaa tacttcaagt cccccttaaa agctatcacc cagtctttgg tgttttttca ccccataata atcactcttt ctcctgtctt tctacaaaga gaaaggagga taagtgtggg gggtttaaag gactggtaaa tttttccaga aatcactact gcattgcact aaacacagct What is the sequence of amino acids encoded by this piece of DNA? But first, we need to know where the translational start site is. There are eight possible initiation codons – which is the one? Important: also need to find out the 5’-end of the mRNA transcript. Human Interleukin-2 (IL-2) gene, exon 1 In Eukaryotes, scanning Open Reading Frame (ORF) is complicated by Introns Effect of Point mutations Effect of Deletion mutations