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Lecture 4: DNA transcription 1) What is the central dogma of molecular biology 2) 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 Central dogma of molecular biology Transcription DNA directed RNA synthesis What is the biological significance? Allows selective expression of genes Regulation of transcription controls time, place and level of protein expression Basic structure of a gene Regulatory region coding region E:\Lessons\5-4_Transc-Translb3\Transc-Transl.swf Transc-Transl.htm Transcription Transcription 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. 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. 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. 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 Where does transcription take place? Transcription in eukaryotes Step 1: transcribing a primary RNA transcript Step 2: modification of this transcript into mRNA Step 1 - overview A.Initiation A) RNA polymerase binds to promoter & opens helix B.Polymerisation B) De novo synthesis using rNTPs as substrate Chain elongation in 5’-3’ direction C) stops at C. Termination termination signal A) Initiation: ENZYME RNA polymerase holoenzyme an agglomeration of many different factors that together direct the synthesis of mRNA on a DNA template Has a natural affinity for DNA Initiation: SIGNAL specific DNA sequences called promoters 1) Region where RNA polymerase binds to initiate transcription 2) Sequence of promoter determines direction of RNA polymerase action 3) Rate of gene transcription depends on rate of formation of stable initiation complexes PROMOTERS Prokaryotes Near 5’ end of operons Pribnow box – consensus sequence TATAAT Fig 29-10: Voet and Voet PROMOTERS Eukaryotes Near 5’ end of genes Recognised by RNA pol II Consensus promoter sequence for constitutive structural genes – GGGCGG Selective structural genes – TATA ENHANCERS Sequences that are associated with a promoter Enhance the activity of a promoter due to its association with proteins called transcription factors Enhancers mediate most selective gene expression in eukaryotes Polymerisation RNA polymerase binds to promoter & opens helix RNA polymerase catalyses addition of rNTPs in the 5’-3’ direction RNA polymerase generates hnRNAs (~70-1000 nt long) & all other RNAs Stops at termination signal Termination specific termination sequence e.g E.coli needs 4-10A followed by a palindromic GC rich region Additional termination proteins e.g. Rho factor in E.coli Step 2: Modification Post transcriptional processing 3 main steps 1) RNA capping, 2) polyadenylation 3) splicing Post transcriptional processing Control of gene expression following transcription but before translation Conversion of primary transcript into mature mRNA Occurs primarily in eukaryotes Localised in nucleus Post transcriptional processing 1) Capping Addition of 7 methylguanosine at 5’ end Mediated by guanylyltransferase Probably protects against degradation Serves as recognition site for ribosomes Transports hnRNA from nucleus to cytoplasm 2) Tailing Addition of poly(A) residues at 3’ end Transcript cleaved 15-20nt past AAUAAA Poly(A)polymerase and cleavage & polyadenylation specificity factor (CPSF) attach poly(A) generated from ATP 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 Cell type-specific splicing Differential splicing in specific tissues Regulation of gene expression Prokaryotes Eukaryotes • Other levels of regulation • Mainly at inlcude posttranscriptional transcriptional level and posttranslational • Sets of genes regulation transcribed together • Each gene transcribed (polycistronic) independently (monocistronic) • E.g. lac operon and trp operon in bacteria RNA polymerase Prokaryotes single multisubunit RNA polymerase complex RNA polymerase Eukaryotes - 3 types exist RNA pol I RNA pol II RNA pol III Located in nucleoli Synthesises most rRNA precursors Located in nucleoplasm Synthesises mRNA precursors Located in nucleoplasm Synthesises 5S rRNA, tRNA, snRNAs RNA polymerase Enzymes that catalyse the formation of RNA using DNA as a template De novo synthesis using rNTP as substrates 1960 – J Hurwitz & S Weiss (RNA)n + rNTP = (RNA)n+1 + Ppi Antibiotics such as Rifampicin / rifamycin B inhibit RNA polymerase activity Gene expression efficiency When to transcribe gene? How many copies to be transcribed? DNA binding proteins Proteins that recognise & bind to specific DNA sequences Recognition determined by specific structural motifs e.g. helix – loop –helix, zinc finger, leucine zipper Examples include Transcription factors • general transcription factors • Upstream transcription factors • Inducible transcription factors Activators Repressors (silencers) How does transcriptional control differ in pro and eukaryotes? Prokaryotes Genes are usually switched ‘on’ by default Repressor proteins needed to ‘stop’ transcription Eukaryotes Genes are usually switched ‘off’ by default Transcriptional activators needed to induce transcription Regulated by chromatin structure, DNA methylation etc Lac operon Fig 29-3/5 : Voet and Voet Fig 8-20: Essential Cell Biology by Alberts et al