L2 Transcription and Processing of Eukaryotic Genes

L2 Transcription and processing of eukaryotic genes Learning outcomes the role of RNA in gene expression during the flow of genetic information the main components of a transcription initiation complex (RNA polymerase II, promoters, general transcription factors) the order in w...

L2 Transcription and processing of eukaryotic genes Learning outcomes the role of RNA in gene expression during the flow of genetic information the main components of a transcription initiation complex (RNA polymerase II, promoters, general transcription factors) the order in which transcription is initiated in eukaryotes models explaining the mechanisms of processing of eukaryotic mRNA during transcription initiation; as well as 3’ end processing and transcription termination. The role of RNA in gene expression during the flow of genetic information the main components of a transcription initiation complex (RNA polymerase II, promoters, general transcription factors) Gene transcription is a modular, coordinated process. Central dogma: DNA -> RNA -> Protein. DNA stored in nucleus and RNA relays information from the nucleus to the cytoplasm. Transcription: DNA base information is copied into RNA. DNA-dependent RNA polymerases transcribe DNA DNA transcription does not need a primer. Transcription proceeds at 33-50 nt/sec. Transcription errs 1 in every 10 000 nucleotides DNA dependent RNA polymerases: RNA polymerase 1 (50%): Ribosomal RNA, used for scaffold of ribosomes and is required for translation. RNA polymerase 2 (3%): Used for mRNA and non-coding RNAs e.g. snRNA/ RNA polymerase 3 (15%): Transfer RNA (mediates transfer of information on ribosomes to protein and some catalytic RNA. Start and end of transcription mRNA is processed prior to export to ribosomes in the cytoplasm Start = promoter End = polyA signal Not all genes are transcribed in every cell There are 21300 protein coding genes though only 5000 per cell are transcribed and processed as not all promotors are active in every cell. Protein-coding pre-mRNA is processed to mRNA by acquiring a cap and a poly(A) tail. The order in which transcription is initiated in eukaryotes models explaining the mechanisms of processing of eukaryotic mRNA during transcription initiation; as well as 3’ end processing and transcription termination. Transcription initiation Transcription initiation depends on proteins binding DNA and Pol II. DNA binding proteins recognise specific regions that are 6-8bp. Through hydrogen-bonds, hydrophobic or electrostatic interactions, weak interactions form. In combination of 10-20, the weak interactions will cause tight binding. Major groove offers more electrostatic or HB interactions than minor Binding of protein bends DNA Consensus sequence: The frequency with which a nucleotide is seen in a binding sequence, indicated by the height of the nucleotide. Promoter: A promoter is the genetic element necessary for expression of a structural gene (or operon) that is distinct from elements that regulate the expression level of the gene Examples of RNA Polymerase II promoters:  BRE = B recognition element INR = initiator element DPE = downstream promoter element Role of TATA binding protein TATA binding protein is required by all polymerases. TBP binds and kinks DNA by 80 degrees, which helps the polymerase find access to DNA and brings elements of the promoter close together. TBP deposition is tightly regulated to prevent unscheduled transcription. Preinitiation complex assembly: 1) TFIID establishes sequence specificity and controls TBP binding. o TFIID consists of TBP and 13 TF2D associated factors. o TFIID binds INR and DPE which delivers TBP to DNA. o TBP displaces Taf1 so TBP can bind DNA. Distance between TBP and TFIID is ~32 bp regardless of sequence. 2) TBP Kinks DNA o 80 degrees 3) TFIIB recruited to TBP-TFIIA dimer and binds to the BRE. 4) TFIIB recruits PolII-TFIIF complex. 5) TFIIH and TFIIE recruited to prevent TFIID rebinding and produce additional bending. 6) TFIIB and XPB increase bending and positive supercoiling, allowing opening up of DNA in active site. 7) TFIIH XPB translocates and this helps opening up DNA. The core promoter sequence/consensus elements are not present in all promotors. TBP can be positioned though other elements with no tata box e.g. SAGA complex. Summary: Initiation requires DNA kinking through TBP, which can be positioned by many factors. Torsional strain is introduced by TBP, TFIIE/B General transcription factors (GTFs) required for transcription on naked DNA, Chromatin context requires more TFs. Transcription elongation Abortive initiation turns into productive elongation when CTD-S5 is phosphorylated o Polymerase terminates transcription elongation at 9-50bp if the necessary transcription factors are not present. o Elongation occurs at 1500-2700bp/min and can be sped up by fast trigger loops. CTD phosphorylation during the transcription cycle coordinates binding of transcription factors o Phosphorylation of S5P on the C terminal domain of RNA polymerase by TFIIH CDK7 overcomes abortive initiation. o Kinases change CTD phosphorylation patterns co-transcriptionally. o RNA processing is coupled to transcription through phospho-CTD- specific binding. o Elongation factors can bind phospho-CTD To prevent premature termination of stalled polymerases as well as facilitate passage through nucleosomes. o Changes in phosphorylation changes the electrostatic surface of the C terminal domain, so changes which proteins can recognise and bind to it. This signals which stage of transcription the polymerase is at. Capping is performed by three enzymatic steps (two polypeptides in humans): removal of phosphate, addition of GTP and methylation of Guanosine. o Capping prevents degradation from 5’ to 3’. Splicing removes non-coding intronic sequences o Introns have GU at the beginning and AG at the end to signal to splicing proteins. o Introns and exons are recognised by sequence specific recognition. Mature, spliced mRNA have the coding sequence (cds) or open reading frame (ORF), in addition to a 5’ and a 3’ untranslated region. o The UTRs help regulate export etc. Transcription termination Termination involves 3’end processing: cleavage and polyadenylation. Factors involved in the endonuclease module bind to the polyA signal, therefore this ensures that the endonuclease is only licenced when the full complex is assembled on RNA. Modal assembly of the large cleavage/poly-adenylation complex is in many ways analogous to the formation of the transcription -initiation complex. After RNA 3’ end processing nascent RNA is degraded by exonuclease Xrn2 degrades approximately 2 kb/min Pol II elongation rate ca 2.7 kb/min Pol II is slowed down through the Spt5 elongation factor Combination of speed control and chasing of RNA Pol II by an exonuclease destabilise the transcription complex Pol II also transcribes non-coding RNA