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Computational Biology I LSM5191 Aylwin Ng, D.Phil Lecture 2 Notes: Molecular Biology of Gene Expression. Flow of information: DNA to polypeptide Start DNA Exon1 Intron Exon2 Termination Transcription Addition of 5’cap m7Gppp Cleavage & addn of polyA tail at 3’end m7Gppp A…(A)200 RNA splicing m7Gppp A…(A)200 Transport to cytoplasm Translation Polypeptide TRANSCRIPTION TRANSCRIPTION – An Overview BACTERIAL GENE EXPRESSION • In prokaryotes, genes encoding proteins involved in related functions often are located next to each other in bacterial chromosomes. • This cluster of genes comprise a single transcription unit called an OPERON. • i.e., a single mRNA molecule contains the full set of genes of the operon. • Hence, prokaryotic mRNA encodes several polypeptides and is therefore polycistronic (a cistron is defined as a genetic unit that encodes a single polypeptide). • Poly-cistronic mRNA contains multiple ribosome-binding sites near start sites for all protein coding regions in the mRNA. BACTERIAL GENE EXPRESSION (cont.d) By convention, the transcription-initiation site in the DNA sequence is designated +1, and base pairs extending in the direction of transcription (downstream) are assigned positive numbers which those extending in the opposite direction (upstream) are assigned negative numbers. Various proteins (RNA polymerase, activators, repressors) interact with DNA at or near the promoter to regulate transcription initiation. E. coli PROMOTER SITES • 2 regions (-10 and –35 regions) in most E. coli promoters are critical for binding RNA polymerase holoenzyme (β’,β,α,α,σ70) via its σ70 subunit (or initiation factor). • After holoenzyme transcribes approx 10 bp, σ70 subunit is released. • The core RNA polymerase (β’,β,α,α) continues transcribing (chain elongation). • ‘Strong’ promoters = promoters at which RNA pol initiates transcription at high frequency (dependent on enzyme’s affinity for promoter). Identification of PROMOTER SITES • DNase I footprinting assays identify protein-DNA interactions. • DNase I randomly hydrolyses phosphodiester bond. • Low concentration of DNase I used Æ on average each DNA molecule is cleaved just once. 3’ G G A T C A EUKARYOTIC TRANSCRIPTION The basic principles that control transcription in bacteria also apply to eukaryotic organisms. Transcription is initiated at a specific base pair and is controlled by the binding of trans-acting proteins (transcription factors) to cis-acting regulatory DNA sequences. However, eukaryotic cis-acting elements are often much further from the promoter they regulate, and transcription from a single promoter may be regulated by binding of multiple transcription factors to alternative control elements. Transcription control sequences can be identified by analysis of a 5′deletion series. Unlike prokaryotes, eukaryotes have 3 (instead of just one) RNA polymerases (all large multi-subunit enzymes). Eukaryotic mRNAs are generally monocistronic. Prokaryotic mRNAs are generally polycistronic, i.e. several polypeptides are translated from the same mRNA. EUKARYOTIC RNA POLYMERASES RNA Polymerase I: • Transcribes gene encoding ribosomal RNA (45S precursor yielding 28S, 18S, 5.8S rRNAs) RNA Polymerase II: • Transcribes all protein-coding genes, • Transcribes genes encoding small nuclear RNAs U1, U2, U3 etc. RNA Polymerase III: • Transcribes genes encoding transfer RNA, • Transcribes gene encoding 5S rRNA, • Transcribes gene encoding snRNA U6. RNA POLYMERASE I • Essential protein factors (rather than the polymerase) recognise DNA sequences around transcription start site. • Key sequences recognised by these factors are located within 50 bases upstream of start site. • SL1 factor recruits RNA polymerase I. UBF UBF = Upstream Binding Factor -50 +1 +50 UBF SL1 -50 +1 +50 UBF SL1 RNA pol I -50 +1 Transcription +50 RNA POLYMERASE II TFIIA TFIID -50 TATA +1 TFIIA TFIID TFIIB -50 TATA RNA pol II +1 TFIIF TFIIA TFIID TFIIB -50 TATA Brief Notes: • TFIIB recruits RNA pol II. • TFIIH phosphorylates C-term domain of RNA pol II. • Phosphorylated form is able to initiate transcription. +1 TFIIETFIIH RNA pol II RNA pol II TFIIF TFIIF TFIIA TFIID TFIIB -50 TATA TFIIA TFIID +1 -50 TATA +1 Transcription RNA POST-TRANSCRIPTIONAL EVENTS in EUKARYOTES Capping Polyadenylation RNA splicing RNA transport Translation CAPPING • Capping only occurs in Eukaryotes! • 5’ end of nascent mRNA is modified, • Addition of a Methylated Guanylate residue (NOT encoded by DNA). • Rxn catalysed by guanylyl transferase. • 3 phosphate molecules separate the G residue from the first nucleotide in the chain (whereas only 1 P separates the other nucleotides). • Guanylate is joined via a 5’-5’ linkage rather than the std. 3’-5’ linkage which links nucleotides in a growing chain. • Cap protects RNA from degradation by 5’Æ3’ exonuclease activity. POLYADENYLATION • Cleavage at 3’ end of mRNA • Addition of poly(A) tail at 3’end of cleaved mRNA Poly(A) site 5’ AAUAAAA CPSF 5’ CPSF: Cleavage & Polyadenylation Specificity Factor CstF AAUAAAA 5’ 3’ G/U 3’ G/U CPSF CstF: Cleavage stimulation factor Endonucleolytic cleavage CstF 3’ CPSF 5’ CstF 3’ AAUAAAA 5’ Poly(A) polymerase 5’ AAUAAAA (A)200 G/U Degradation 3’ 3’ Role of polyadenylation • To protect mRNA from degradation by exonucleases. • Exonucleases ‘attack’ its free 3’ end and rapidly degrades mRNA. • Appears to increase the efficiency by which an mRNA is translated. Not all mRNAs (encoding proteins) are polyadenylated, e.g.mRNAs encoding Histones. • mRNA fold itself into a double-stranded stem-loop structure which protects it from degradation. EXONS & INTRONS • Protein-coding regions of a gene are known as EXONS. • Intervening regions that do not encode parts of protein are known as INTRONS. • Introns are transcribed into mRNA, but remains in nucleus. • Hence, primary RNA transcript must have its introns removed before being transported into the cytoplasm and translated. • RNA SPLICING is the process whereby introns are removed. RNA SPLICING – what’s the mechanism? • Clue: Short, conserved sequences at splice junctions. RNA SPLICING 5’ splice site 5’ 3’ splice site A GU Exon 1 AG Intron 3’ Exon 2 Cleavage at 5’ splice site 5’ 3’ 5’ Exon 1 5’ 3’ U G 5’ 2’A U G 5’ 2’A 3’ AG Exon 2 Exon 1 3’ Cleavage at 3’ spliced site AG Intron Exon 1 3’ Exon 2 Intron Exon 1 5’ AG Intron Lariat formation 5’ A GU Exon 2 3’ 5’ Exon 2 3’ 3’ In vitro analysis (Ruskin et al., 1984) Nuclear extract (from cells) incubated with radio-labelled RNA: Starting RNA Final spliced product Excised intron Small nuclear RNAs (snRNAs) • These are splicing ‘factors’, i.e. assist in the splicing process. • NOTE: they are NOT proteins, but RNA molecules !!! • But snRNAs associate with small nuclear ribonucleoproteins (snRNPs) to form a large ribonucleoprotein complex called a Spliceosome. SPLICEOSOMES assembly ALTERNATIVE SPLICING • A mechanism for tissue-specific expression • E.g. Hepatocytes generate fibronectin proteins that are different from those produced by Fibroblasts. RNA TRANSPORT • Spliced mRNA must be transported out from the nucleus (across the nuclear membrane) into the cytoplasm for translation into protein. • Heterogeneous nuclear RNPs (hnRNPs) is likely to mediate this transport by associating with mRNA in nucleus. • In yeast, the Gle1 protein mediates this transport. • The Gle1 protein contains a short nuclear export signal (NES) sequence. • NES sequence is also present in HIV’s Rev protein, which is involved in regulating the nuclear-cytoplasmic transport of different HIV mRNAs. TRANSLATION RIBOSOMES • 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 protein synthesis (translation): Messenger RNA (mRNA) carries information copied from DNA in the form of a series of three base “words” termed codons Transfer RNA (tRNA) deciphers the code and delivers the specified amino acid Ribosomal RNA (rRNA) associates with a set of proteins to form ribosomes, structures that function as protein-synthesizing machines TRANSFER RNA (tRNA) • tRNA forms the vital link between mRNA & the growing polypeptide chain. • 50 different tRNAs in eukaryotes. • But only 20 amino acids are designated by the genetic code. Æ different tRNAs (isoacceptors) are specific for the same amino-acid (due to ‘wobble’ base-pairing). • Nomenclature: e.g. tRNAGly1 and tRNAGly2 are both specific for glycine. • Amino-acid is attached at 3’-end of tRNA. • All mature tRNA ends with –CCA. (CCA added by tRNA nucleotidyl-transferase). • Anti-codon base-pairs with CODON on mRNA during translation. What is a Codon? • A unit of 3 nucleotides. • An amino acid is encoded by a Codon (except for stop codons). TRANSFER RNA (tRNA) Cloverleaf structure (dihydrouridine) (ThymidinepseudoU-cytidine) 3-D Structure Aminoacylation (‘charging’) of tRNA • • • • Attachment of amino-acid (a.a) to tRNA. Enzymes req.d: 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 tRNA 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’ results in non-standard base pairing: • 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 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 info: also need to know where the transcription start site is. 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