Presentation on theme: "Lecture 4: DNA transcription"— Presentation transcript:
1 Lecture 4: DNA transcription 1) What is the central dogma of molecular biology2) What are the steps involved in transcribing a primary RNA transcript?3) How does eukaryotic post-transcriptional processing convert a primary transcript into messenger RNA?4) Write notes on promoters, enhancers and transcription factors
6 TranscriptionTranscription is the mechanism by which a template strand of DNA is utilized by specific RNA polymerases to generate one of the three different types of RNA.
7 Types of RNA 1) Messenger RNA (mRNA) This class of RNAs are the genetic coding templates used by the translational machinery to determine the order of amino acids incorporated into an elongating polypeptide in the process of translation.
8 Types of RNA….. 2) Transfer RNA (tRNA) This class of small RNAs form covalent attachments to individual amino acids and recognize the encoded sequences of the mRNAs to allow correct insertion of amino acids into the elongating polypeptide chain.
9 Types of RNA….. 3) Ribosomal RNA (rRNA) This class of RNAs are assembled, together with numerous ribosomal proteins, to form the ribosomes. Ribosomes engage the mRNAs and form a catalytic domain into which the tRNAs enter with their attached amino acids. The proteins of the ribosomes catalyze all of the functions of polypeptide synthesis
11 Transcription in eukaryotes Step 1: transcribing a primary RNA transcriptStep 2: modification of this transcript into mRNA
12 Step 1 - overview Initiation Polymerisation C. Termination A) RNA polymerase binds to promoter & opens helixB) De novo synthesis using rNTPs as substrateChain elongation in 5’-3’ directionC) stops at termination signal
13 A) Initiation: ENZYME RNA polymerase holoenzyme an agglomeration of many different factors that together direct the synthesis of mRNA on a DNA templateHas a natural affinity for DNA
14 Initiation: SIGNAL specific DNA sequences called promoters 1) Region where RNA polymerase binds to initiate transcription2) Sequence of promoter determines direction of RNA polymerase action3) Rate of gene transcription depends on rate of formation of stable initiation complexes
15 PROMOTERS Prokaryotes Fig 29-10: Voet and Voet Near 5’ end of operons Pribnow box – consensus sequence TATAATFig 29-10: Voet and Voet
16 PROMOTERS Eukaryotes Near 5’ end of genes Recognised by RNA pol II Consensus promoter sequence forconstitutive structural genes – GGGCGGSelective structural genes – TATA
17 ENHANCERS Sequences that are associated with a promoter Enhance the activity of a promoter due to its association with proteins called transcription factorsEnhancers mediate most selective gene expression in eukaryotes
18 Polymerisation RNA polymerase binds to promoter & opens helix RNA polymerase catalyses addition of rNTPs in the 5’-3’ directionRNA polymerase generates hnRNAs (~ nt long) & all other RNAsStops at termination signal
19 Termination specific termination sequence e.g E.coli needs 4-10A followed by a palindromic GC rich regionAdditional termination proteinse.g. Rho factor in E.coli
20 Step 2: Modification Post transcriptional processing 3 main steps RNA capping,polyadenylationsplicing
21 Post transcriptional processing Control of gene expression following transcription but before translationConversion of primary transcript into mature mRNAOccurs primarily in eukaryotesLocalised in nucleus
23 1) Capping Addition of 7 methylguanosine at 5’ end Mediated by guanylyltransferaseProbably protects against degradationServes as recognition site for ribosomesTransports hnRNA from nucleus to cytoplasm
24 2) Tailing Addition of poly(A) residues at 3’ end Transcript cleaved 15-20nt past AAUAAAPoly(A)polymerase and cleavage & polyadenylation specificity factor (CPSF) attach poly(A) generated from ATP
25 3) Splicing Highly precise removal of intron sequences Performed by spliceosomes (large RNA-protein complex made of small nuclear ribonucleoproteins)Recognise exon-intron boundaries and splice exons together by transesterification reactions
26 Cell type-specific splicing Differential splicing in specific tissues
27 Regulation of gene expression ProkaryotesMainly at transcriptional levelSets of genes transcribed together (polycistronic)E.g. lac operon and trp operon in bacteriaEukaryotesOther levels of regulation inlcude posttranscriptional and posttranslational regulationEach gene transcribed independently (monocistronic)
29 RNA polymerase Eukaryotes - 3 types exist RNA pol I RNA pol II RNA pol IIILocated in nucleoliLocated in nucleoplasmSynthesises most rRNA precursorsSynthesises mRNA precursorsSynthesises 5S rRNA, tRNA, snRNAs
30 (RNA)n + rNTP = (RNA)n+1 + Ppi RNA polymeraseEnzymes that catalyse the formation of RNA using DNA as a templateDe novo synthesis using rNTP as substrates1960 – J Hurwitz & S Weiss(RNA)n + rNTP = (RNA)n+1 + PpiAntibiotics such as Rifampicin / rifamycin B inhibit RNA polymerase activity
31 Gene expression efficiency When to transcribe gene?How many copies to be transcribed?
32 DNA binding proteins Examples include Transcription factors Proteins that recognise & bind to specific DNA sequencesRecognition determined by specific structural motifse.g. helix – loop –helix, zinc finger, leucine zipperExamples includeTranscription factorsgeneral transcription factorsUpstream transcription factorsInducible transcription factorsActivatorsRepressors (silencers)
33 How does transcriptional control differ in pro and eukaryotes? ProkaryotesGenes are usually switched ‘on’ by defaultRepressor proteins needed to ‘stop’ transcriptionEukaryotesGenes are usually switched ‘off’ by defaultTranscriptional activators needed to induce transcriptionRegulated by chromatin structure, DNA methylation etc