Chapter 12 and 13: Transcription and Translation Lecture 12 October 28, 2003

1. 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

2. •Structural analysis of DNA 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 *Composed of nucleotides – deoxyribose phosphate group nitrogenous base *Strands are antiparallel and complementary A – T C - G

3. •Semiconservative - each DNA molecule consists of one parental and one newly synthesized strand Review: Molecular Basis of Genetics, so far… Replication •Mode of DNA Replication *Meselson and Stahl – “heavy” and “light” nitrogen isotopes •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

4. 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

5. Also... TEXT: A DNA sequence coding for a single polypeptide 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 KSM: A DNA sequence that produces a functional RNA molecule *Non (protein) coding RNA’s

6. Phenotype 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

7. RNA Transcript 5’ 3’ terminus +1 start site 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) *Un-translated regions (UTR’s) Promoter regions – sequence involved in the control of expression of a given gene, site where RNA polymerase binds Gene: A Molecular Description In eukaryotes: introns and exons Non-coding regions – contains nucleotide sequence that will get transcribed BUT not translated Regulatory regions – sequence involved in the control of expression of a given gene, usually involves interaction with another molecule

8. Coding strand 5’ 3’ A A A G T C C G G T A C G T T T C A G G C C A T G C 3’ 5’ U U U C A G G C C A U G C 3’ 5’ 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 Given that RNA polymerase synthesizes RNA in a 5’ to 3’ direction, which strand is the template strand? *Transcript will always “look” like the non-sense strand

9. Sigma factor – helps drive the polymerase to the promotor s a2b b’ Core – responsible for elongation Holoenzyme responsible for initiation = binding of the polymerase to the promotor Sigma factor Core Holoenzyme 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 )

10. RNA Transcript +1 start site P1 s1 Coding Region P2 s2 P3 s3 5’ 3’ 3’ 5’ TTGACA -10, TATA box, Pribnow box TATAAT ~17 base spacer -35 region Transcription Factor – something that cycles on and off core complexes Multiple types of sigma factors in bacterial cells - regulation Promotors - sequence involved in the control of expression of a given gene, site where RNA polymerase binds Serve three different functions: 1. ON/OFF switch 2. “Speed” switch 3. Alignment

11. Hogness box CAAT box Hogness box CAAT box Hogness box CAAT box Hogness box Goldberg-Hogness box, TATA box, -25 (all) CAAT box, -80 (many) 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: Enhancers

12. 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

13. 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