2 Remember the Central Dogma A copy of the DNADNAInformation in sequence of basesRNAIntermediate ‘messenger’ - mRNAProteinSequence of amino acidsThe things that do the work in a cell.Only certain bits of the DNA copiedMultiple copies madeAllows temporal and physical separation between DNA and protein manufactureWhy don’t we just make the proteins directly from the DNA?
3 Procaryotic vs Eucaryotic We’ll start with ProcaryoticSimpler, no nucleusAlso control much simplerControl is the most important and exciting thingEach of the cells in your body contains all the information to make youBut 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 cellsThe cells will also express many genes in commonMetabolic pathways, cell receptors, membrane assembly, etc
4 Genotype and Phenotype Your DNA determines your GENOTYPEWhat’s expressed determines your PHENOTYPEThe proteins that are madeGENOME vs PROTEOMEThe time-course of expression is also importantDuring development and differentiation of cellseg, Embryonic developmentGenes must be switched on and off in the correct orderThe 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 DNAbut using ribonucleotides instead of deoxyribonucleotidesLinked by phosphoester bonds between the 5’ residue of one nucleotide and the 3’ residue of anotherUsing uracil in place of thymineMade using instruction from DNA templateUsing RNA Polymerase
7 Basic Reaction Then new triphosphonucleotide comes in Phosphoester bonds formedRelease of pyrophosphateSpontaneous hydrolysis of PP pulls reaction to completionNote the directionality5’ to 3’And the lack of primerBut does need the DNA templateNew chain is anti-parallel to the DNASo the DNA template is read 3’ to 5’5’3’PPPOH5’5’3’3’PPPPPPPOHOH+PP
8 Starting Transcription How does RNA pol know where to start?DNA is long!Specific sequences that signify the beginning of a geneThe PROMOTER regionPromoter regionThe gene – the bit that will be copied into mRNAUPSTREAM-1+1DOWNSTREAM
9 The Promoter Contains a consensus sequence Not all promoters have this exact sequenceBut the nearer they are to it, the more strongly the gene is initiatedIt can be on either DNA strandWhichever one it is on, the opposite one that is transcribedThe TEMPLATE strandBoth strands of DNA can act as the template in different sectionsBoth strands contain genesPromoter region specifies the site and direction of mRNA synthesis------TTGACA TATAAT |-------35-10UPSTREAM-1+1DOWNSTREAM
10 RNA Polymerase Four main subunits in the CORE enzyme α2β’βBeta catalyses the polymerisationBeta prime keeps the enzyme on trackAlphas can associate with other proteinsTo initiate RNA pol has to have a partnerσ, the sigma subunitCan find the promoter sequence, even though the DNA is double strandedWith sigma, RNA pol is called the HOLOENZYMESigma binds to promoter regions with 10 million times the affinity than random DNASpecific 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 promoterOpen area initially about 80 basesBetween -55 and +20Assisted by high A=T content in this regionNegative super-coiling also helps unwind the DNABeta subunit catalyses the first nucleotide entryUsually a purine (G or A)A triphosphonucleotideRNA polσ-55+20
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 downSigma falls offRemember it likes ds DNA, not ss DNA or DNA/RNA hybridSigma now free to do more initiatingRNA starts to peel off template strandNusA binds to RNA polHelps keep it on trackIt doesn’t take much to put RNA pol off track!!
13 Elongation Elongation rate about 40 nucleotides/sec NO PROOF READING Average gene takes about 20 seconds to transcribeNO PROOF READINGUnlike DNA pol, RNA pol has no 3’ to 5’ exonuclease activity1 mistake in 10,000 nucleotides
14 Termination We only want a small portion of DNA copied! Two ways of stoppingFactor independentDepends on the shape of the mRNA that’s formedRNA pol pauses when these structures formedRemember how easy RNA pol is to knock off its tracksFactor dependentRequires a protein factor that chases RNA polRho, ρA circular hexamer of six identical subunits that encloses the single stranded RNA and hydrolyses ATP as it zooms in towards the transcription bubbleUnwinds the DNA-RNA hybrid and kicks off RNA polEspecially when the latter has paused
15 Factor Independent Termination mRNA can form intra-molecular base-pairsGGGGGGGGG-----CCCCCCCC UUUUUUUUU-3’OHGCUUUUUUUUU-3’OHAs the hairpin loop forms, the mRNA is pulled off the DNAThis rather weak tail helps!
16 Extras… and CistronsIf the transcription bubble gets out of the way quickly, re-initiation occurs rapidlyPROMOTER CLEARANCEThousands of copies can be made after initiationSo the odd mistake doesn’t matterRNA is VERY labileCistronsA stretch of mRNA that contains structural informationOften bacterial messages are POLYCISTRONICEach mRNA contains multiple stop/start sites for multiple genes3’ and 5’ untranslated regions (UTRs)Translation does not start or finish right at the ends of the mRNAContain information relevant to gene stability, etc
17 Textbook Refs Chapter 8 Clarke Chapter 6 All the introductory blurb All the sections on the Enzymatic Synthesis of RNA (p )Transcription signals on p150But NOT the intimate structure of the sigma subunitAlthough figure 8-4 is beautifulThe figure showing different promoter regions obviously doesn’t need to be memorisedAll of p151Except the details of nucelotide binding sites in RNA polAll of p152 and 153Although the diagram on p153 makes things look complicatedp154But not the stuff on the classes of RNAClarke Chapter 6Everything from p133 to p140
18 Advanced Only DNA footprinting To study the interaction between protein and DNASpecifically the sequences on the DNA that bind to the proteinFind out which sequences are protected from digestionOriginal referenceDNAase footprinting: Galas & Schmitz (1978) Nucleic Acids Research 5 (9) 3157
19 In the original paper, done with lac repressor Fragment of double stranded DNALabeled with radioactivity at one endRun the fragments on a gelReveal the radioactively labeled strands using x-ray filmLadder produced. Smaller fragments run fasterThousands of copies of the fragmentDenature the double strandsIncubate with DNAaseINow do the same with the digestion step in the presence of the DNA binding protein.Each copy cut in a different placeEvery possible length represented