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Chapter 12 and 13: Transcription and Translation Lecture 12 October 28, 2003 What’s due? CH6 and CH10 problem set (if you haven’t all ready turned it in) CH 11 problem set Review: Molecular Basis of Genetics, so far… Structure •DNA as the genetic material *Griffith – “transforming principle” *Avery, MacLeod and McCarty - DNA was the “transforming principle” *Hershey and Chase - DNA was the genetic material •Structural analysis of DNA *Composed of nucleotides – deoxyribose phosphate group nitrogenous base *Strands are antiparallel and complementary A – T C - G Review: Molecular Basis of Genetics, so far… Replication •Mode of DNA Replication *Meselson and Stahl – “heavy” and “light” nitrogen isotopes •Semiconservative - each DNA molecule consists of one parental and one newly synthesized strand •Origin of replication •Bi-directional •Roles of each polymerase (prokaryotes): DNA polymerase I - primer removal, gap-filling synthesis DNA polymerase II - DNA repair DNA polymerase III - main replication enzyme •At least six DNA polymerases in eukaryotes Review: A Coherent Model of DNA Replication •Helicases unwind helix (DnaA, B and C) •SSBPs prevent closure •DNA gyrase reduces tension •Association of core polymerase with template •Primase synthesizes short RNA primer •DNA synthesis (DNA pol III) •Primer removal and replacement with DNA (DNA pol I) •Ligase closes up the gaps b/w Okazaki fragments Gene Expression: Transcription and Translation Gene expression – mechanism by which hereditary factors are coded for and expressed (“to cause a gene to manifest its effects in the phenotype” or “the detectable effect of a gene”) Gene – unit of inheritance which occupies a specific chromosomal location Also... TEXT: A DNA sequence coding for a single polypeptide KSM: A DNA sequence that produces a functional RNA molecule Type of RNA Encodes Copies/genome mRNA Functional protein Single or few tRNA Molecule needed for translation Few rRNA Component of ribosomes Many *Non (protein) coding RNA’s Gene Expression Protein coding gene - A DNA sequence coding for a single polypeptide Gene expression – mechanism by which hereditary factors are coded for and expressed Genes control inherited variation via: DNA, RNA and protein *Transcription – transfer of genetic information from DNA via synthesis of RNA *Translation– the formation of a protein, directed by an mRNA in association with a ribosome Phenotype Gene: A Molecular Description RNA Transcript terminus +1 start site 5’ 3’ 3’ 5’ Coding Region Promoter 5’ UTR 3’ UTR Coding region – contains nucleotide sequence that encodes a specific protein product (this region will be translated) In eukaryotes: introns and exons Non-coding regions – contains nucleotide sequence that will get transcribed BUT not translated *Un-translated regions (UTR’s) Promoter regions – sequence involved in the control of expression of a given gene, site where RNA polymerase binds Regulatory regions – sequence involved in the control of expression of a given gene, usually involves interaction with another molecule Gene: A Molecular Description Only one of the two strands encodes the mRNA for a given gene Template strand – coding strand – sense strand = template for transcription Non-template strand – nonsense strand = RNA transcript is exactly the same as the non-sense strand 5’ A A A G T C C G G T A C G 3’ 3’ T Coding strand T T C A G G C C A T G C 5’ Given that RNA polymerase synthesizes RNA in a 5’ to 3’ direction, which strand is the template strand? 3’ U U U C A G G C C A U G C 5’ *Transcript will always “look” like the non-sense strand Transcription Transcription – the process by which RNA molecules are synthesized on a DNA template *RNA polymerase – enzyme that copies template strand to build an RNA molecule -synthesis in 5’ to 3’ direction –nucleotides added to 3’-OH –growing strand has base complementarity to template strand –unlike DNA pol, no primer required reminder: RNA contains ribose, phosphate group and A, C, G and U (not T) *RNA polymerase (from E. coli ) s a2 b b’ Sigma factor Core Holoenzyme Sigma factor – helps drive the polymerase to the promotor Core – responsible for elongation Holoenzyme responsible for initiation = binding of the polymerase to the promotor Transcription Factor – something that cycles on and off core complexes Multiple types of sigma factors in bacterial cells - regulation s1 P1 s2 P2 s3 P3 Promotors - sequence involved in the control of expression of a given gene, site where RNA polymerase binds +1 start site 5’ 3’ TTGACA TATAAT -35 region RNA Transcript Coding Region -10, TATA box, Pribnow box ~17 base spacer Serve three different functions: 1. ON/OFF switch 2. “Speed” switch 3. Alignment 3’ 5’ Transcription in Eukaryotes *RNA polymerases: RNA polymerase I – rRNA (18S, 28S) RNA polymerase II – mRNA RNA polymerase III – small RNA’s ( tRNA, 5S rRNA, snRNA’s) Eukaryotic promotors: CAAT box Hogness box CAAT box Hogness box Hogness box CAAT box Goldberg-Hogness box, TATA box, -25 (all) CAAT box, -80 (many) Enhancers Hogness box Transcription in Eukaryotes “Generalized Transcription Factors”–group of proteins that bind the -25 region Transcription Factor for RNA polymerase II – TFIIA, TFIIB, etc. *TFII’s – not enough! Need factors that bind -80 and enhancers 1. ON/OFF switch = -25 region 2. “Speed” switch = enhancers Elongation – very similar in prokaryotes and eukaryotes Termination -Transcription stops - Polymerase and RNA are released from DNA - DNA rehybdridizes RNA processing in Eukaryotes Immature RNA – mature RNA *Addition of a cap at 5’ end- guanyltransferase – makes mRNA more stable, required for translation *Addition of a poly A tail – poly A polymerase – mRNA stability, translation *Introns spliced out by spliceosome machinery