Computational Biology I LSM5191
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1 Computational Biology I LSM5191 Aylwin Ng, D.Phil Lecture Notes: Transcriptome: Molecular Biology of Gene Expression II
2 TRANSLATION
3 RIBOSOMES: protein synthesizing machines Translation takes place on defined cytoplasmic organelles called RIBOSOMES.
4 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
5 What is a Codon? A unit of 3 nucleotides. Each Codon (except for stop codons) encodes an amino acid.
6 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.: trna Met carries only methione; and trna Gly 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. trna Gly1 and trna Gly2 are both specific for glycine. All mature trna ends with CCA. (CCA added by trna nucleotidyl-transferase).
7 TRANSFER RNA (trna) Cloverleaf structure 3-D Structure (dihydrouridine) (ThymidinepseudoU-cytidine)
8 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 1 Step 2 Step 2: a.a transferred to 3 -end of trna. Overall rxn: enz a.a + ATP + trna Aminoacyl-tRNA + AMP + 2Pi
9 Codon & trna anticodon recognition Specificity of aminoacylation 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.
10 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.
11 THE GENETIC CODE
12 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 ) initiation of translation. Initiator trna is charged with methionine.
13 TRANSLATION INITIATION (Eukaryotes) Cap 5 AUG (A) n 3 eif4e AUG (A) n 3 eif4a eif4f complex eif4g eif4e AUG (A) n 3 trna eif2 trna eif2 eif4f complex eif4a 40S 40S eif4g eif4e AUG (A) n 3 eif2 60S 40S AUG Translation (A) n 3
14 ELONGATION OF TRANSLATION Mechanism very similar in bacteria and eukaryotes. Peptide bond formation catalysed by peptidyl transferase eef-1
15 Animation clip
16 EXERCISES
17 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)
18 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.
19 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 (4) 5 - A G U 3 (2) and (4) Which amino acid is the trna likely to be specific for? Serine
20 Locating genes by scanning Open Reading Frame (ORF)
21 Human Interleukin-2 (IL-2) gene - promoter, exon 1 and partial cds (Accession Number: AF031845) 1 tatgacaaag aaaattttct gagttacttt tgtatcccca cccccttaaa gaaaggagga 61 aaaactgttt catacagaag gcgttaattg catgaattag agctatcacc taagtgtggg 121 ctaatgtaac aaagagggat ttcacctaca tccattcagt cagtctttgg gggtttaaag 181 aaattccaaa gagtcatcag aagaggaaaa atgaaggtaa tgttttttca gactggtaaa 241 gtctttgaaa atatgtgtaa tatgtaaaac attttgacac ccccataata tttttccaga 301 attaacagta taaattgcat ctcttgttca agagttccct atcactcttt aatcactact 361 cacagtaacc tcaactcctg ccacaatgta caggatgcaa ctcctgtctt gcattgcact 421 aagtcttgca cttgtcacaa acagtgcacc tacttcaagt tctacaaaga aaacacagct 481 acaactggag catttactgc 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.
22 Human Interleukin-2 (IL-2) gene, exon 1
23 In Eukaryotes, scanning Open Reading Frame (ORF) is complicated by Introns
24 Effect of Point mutations
25 Effect of Deletion mutations
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