Transcription

1. Transcription Lecture 11

2. Remember the Central Dogma A copy of the DNA DNA Information in sequence of bases RNA Intermediate ‘messenger’ - mRNA Protein Sequence of amino acids The things that do the work in a cell. Only certain bits of the DNA copied Multiple copies made Allows temporal and physical separation between DNA and protein manufacture Why don’t we just make the proteins directly from the DNA?

3. Procaryotic vs Eucaryotic • We’ll start with Procaryotic • Simpler, no nucleus • Also control much simpler • Control is the most important and exciting thing • Each of the cells in your body contains all the information to make you • But only certain genes are transcribed and translated in specific cells – We call this EXPRESSION. • Muscle cells make the proteins that make them muscle cells (eg, the contractile filaments) • Kidney cells make the proteins that make them kidney cells (their shape and function is completely different) • Yet every cell has the DNA (the ‘genes’) required for these cells • The cells will also express many genes in common • Metabolic pathways, cell receptors, membrane assembly, etc

4. Genotype and Phenotype • Your DNA determines your GENOTYPE • What’s expressed determines your PHENOTYPE • The proteins that are made • GENOME vs PROTEOME • The time-course of expression is also important • During development and differentiation of cells • eg, Embryonic development • Genes must be switched on and off in the correct order • The problem for Jurassic Park was not getting the dinosaur sequence but expressing the genes in the right order!

5. What is mRNA? • A polynucleotide • Like DNA • but using ribonucleotides instead of deoxyribonucleotides • Linked by phosphoester bonds between the 5’ residue of one nucleotide and the 3’ residue of another • Using uracil in place of thymine • Made using instruction from DNA template • Using RNA Polymerase

6. Drawings 5’ PPP OH 3’ OH 1’ N 5’ 3’ PPP OH P P P P P P P

7. Basic Reaction • Then new triphosphonucleotide comes in • Phosphoester bonds formed • Release of pyrophosphate • Spontaneous hydrolysis of PP pulls reaction to completion • Note the directionality • 5’ to 3’ • And the lack of primer • But does need the DNA template • New chain is anti-parallel to the DNA • So the DNA template is read 3’ to 5’ 5’ 3’ PPP OH 5’ 5’ 3’ 3’ PPP P PPP OH OH + PP

8. Starting Transcription • How does RNA pol know where to start? • DNA is long! • Specific sequences that signify the beginning of a gene • The PROMOTER region Promoter region The gene – the bit that will be copied into mRNA UPSTREAM -1 DOWNSTREAM +1

9. The Promoter • Contains a consensus sequence • Not all promoters have this exact sequence • But the nearer they are to it, the more strongly the gene is initiated • It can be on either DNA strand • Whichever one it is on, the opposite one that is transcribed • The TEMPLATE strand • Both strands of DNA can act as the template in different sections • Both strands contain genes • Promoter region specifies the site and direction of mRNA synthesis ------TTGACA---------------------TATAAT----------|------ -35 -10 UPSTREAM -1 +1 DOWNSTREAM

10. RNA Polymerase • Four main subunits in the CORE enzyme • α2β’β • Beta catalyses the polymerisation • Beta prime keeps the enzyme on track • Alphas can associate with other proteins • To initiate RNA pol has to have a partner • σ, the sigma subunit • Can find the promoter sequence, even though the DNA is double stranded • With sigma, RNA pol is called the HOLOENZYME • Sigma binds to promoter regions with 10 million times the affinity than random DNA • Specific for ds DNA (whereas core likes ss DNA better!)

11. Transcription Bubble • Sigma not only finds the right spot • Also helps ‘melt’ open the DNA around the promoter • Open area initially about 80 bases • Between -55 and +20 • Assisted by high A=T content in this region • Negative super-coiling also helps unwind the DNA • Beta subunit catalyses the first nucleotide entry • Usually a purine (G or A) • A triphosphonucleotide σ RNA pol +20 -55

12. Elongation • As each nucleotide comes in • The bubble opens ahead (four nucleotides) • And closes behind (ten nucleotides open) • Once >6 nucleotides have been laid down • Sigma falls off • Remember it likes ds DNA, not ss DNA or DNA/RNA hybrid • Sigma now free to do more initiating • RNA starts to peel off template strand • NusA binds to RNA pol • Helps keep it on track • It doesn’t take much to put RNA pol off track!!

13. Elongation • Elongation rate about 40 nucleotides/sec • Average gene takes about 20 seconds to transcribe • NO PROOF READING • Unlike DNA pol, RNA pol has no 3’ to 5’ exonuclease activity • 1 mistake in 10,000 nucleotides

14. Termination • We only want a small portion of DNA copied! • Two ways of stopping • Factor independent • Depends on the shape of the mRNA that’s formed • RNA pol pauses when these structures formed • Remember how easy RNA pol is to knock off its tracks • Factor dependent • Requires a protein factor that chases RNA pol • Rho, ρ • A circular hexamer of six identical subunits that encloses the single stranded RNA and hydrolyses ATP as it zooms in towards the transcription bubble • Unwinds the DNA-RNA hybrid and kicks off RNA pol • Especially when the latter has paused

15. G G G G G G G C C C C C C C UUUUUUUUU-3’OH Factor Independent Termination mRNA can form intra-molecular base-pairs -----------GGGGGGGGG-----CCCCCCCC-------------UUUUUUUUU-3’OH As the hairpin loop forms, the mRNA is pulled off the DNA This rather weak tail helps!

16. Extras… and Cistrons • If the transcription bubble gets out of the way quickly, re-initiation occurs rapidly • PROMOTER CLEARANCE • Thousands of copies can be made after initiation • So the odd mistake doesn’t matter • RNA is VERY labile • Cistrons • A stretch of mRNA that contains structural information • Often bacterial messages are POLYCISTRONIC • Each mRNA contains multiple stop/start sites for multiple genes • 3’ and 5’ untranslated regions (UTRs) • Translation does not start or finish right at the ends of the mRNA • Contain information relevant to gene stability, etc

17. Textbook Refs • Chapter 8 • All the introductory blurb • All the sections on the Enzymatic Synthesis of RNA (p147-149) • Transcription signals on p150 • But NOT the intimate structure of the sigma subunit • Although figure 8-4 is beautiful • The figure showing different promoter regions obviously doesn’t need to be memorised • All of p151 • Except the details of nucelotide binding sites in RNA pol • All of p152 and 153 • Although the diagram on p153 makes things look complicated • p154 • But not the stuff on the classes of RNA • Clarke Chapter 6 • Everything from p133 to p140

18. Advanced Only • DNA footprinting • To study the interaction between protein and DNA • Specifically the sequences on the DNA that bind to the protein • Find out which sequences are protected from digestion • Original reference DNAase footprinting: Galas & Schmitz (1978) Nucleic Acids Research 5 (9) 3157

19. In the original paper, done with lac repressor Fragment of double stranded DNA Labeled with radioactivity at one end Run the fragments on a gel Reveal the radioactively labeled strands using x-ray film Ladder produced. Smaller fragments run faster Thousands of copies of the fragment Denature the double strands Incubate with DNAaseI Now do the same with the digestion step in the presence of the DNA binding protein. Each copy cut in a different place Every possible length represented