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Download PG1005 Lecture 17 Gene Transcription
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PG1005 Gene Transcription Lecture 17 Dr Neil Docherty My Teaching Objectives • To summarise the gene-RNA-protein central dogma of Gene expression • To outline the various types of RNA molecule found within the cell • To explain the mechanism of action of RNA polymerase II • To describe the key steps involved in mRNA maturation • To introduce the concept of transcriptional regulation Genetic Information Directs Protein Synthesis From Messenger RNA 5’ Gene code (DNA) 3’ 3’ 5’ RNA polymerase II Intermediate (mRNA) 5’ TRANSCRIPTION 3’ NUCLEUS CYTOPLASM ribosome (+rRNAs) membrane rER-Golgi TRANSLATION /TARGETTING organelle secretory vesicle tRNA (+ amino acid) free ribosomes cytoplasmic protein RNA Structure -Linear polymer composed of 4 nucleotide subunits --Nucleotides are ribonucleotides (ribose sugar) -AGCU (adenine, guanine, cytosine and uracil) -Intramolecular complimentary sequences found in RNA Can form intramolecular bonds permitting folding and generation of precise 3D structures RNA DNA Principal Type of RNA (1) There are 5 types you need to know about All have a role in the control of gene expression Not all are intermediate “central dogma” codes however 1) mRNA -Messenger RNA (<2000bp) is the direct coding intermediate for the production of proteins. Passes through various steps of processing before mature form is translated on the ribosome 2) rRNA -ribosmal RNA forms complex with multimolecular protein machinery to form the ribosome. Central to some of the processing steps involved in the production of mature mRNA Principal Type of RNA-2 3) tRNA -Transfer RNAs couple complimentary base pairing with mRNA to the delivery of specific amino acids to the ribosome to provide each monomer of the polypeptide chain 4) snRNA -Small nuclear RNAs, in association with proteins form small ribonucleoprotein particles are involved in the spliceosome which removes stretches of non-coding sequence from immature mRNA 5) miRNA -Small transcripts which are cleaved intracellularly to give rise to short sequences of complimentary RNA which act in concert with protein complexes to directly degrade target mRNAs and/or inhibit translation RNA Polymerases • RNA polymerase I Synthesizes a 45S rRNA which is cleaved to give rise to the 28S, 18S and 5.8S rRNAs, all components of the ribosome • RNA polymerase II Synthesizes mRNA, snRNA and miRNA. • RNA polymerase III Synthesizes tRNA and 5S rRNA Stages of Transcription Three steps1) Initiation of transcription at start site 2) Elongation of nascent RNA 3) Termination of transcription Transcriptional Initiation (RNApol II) 1) Sequential assembly of general transcription factors around the TATA box TBP-TATA binding protein TAF-TBP associated factors N.B. Binding of TBP leads to a pronounced bend in the DNA. 2) TFIIB binds at consensus sites around the TATA box and directs entry of the polymerase 3)TFIIH binds and exercises 2 key functions 1. Helicase activity 2. Kinase activity The pre-initiation complex The Mediator Complex Helps RNApol II to locate at promoters and helps secure assembly Elongation New nucleotides are added to the free 3’ OH end of the growing immature mRNA 5’ Capping Once nascent mRNAs have reached 25bp in length They are capped at the 5’ end by The addition of methylated guanosine nucleotide Termination Introns and Exons • Newly synthesised mRNA is immature and contains transcribed regions of spacer DNA (introns) and protein coding DNA (exons) • As part of maturation the introns are spliced out The Spliceosome Intron recognition and catalysis driven by snRNA in complex with protein (snRNPs) Export of mRNA • Binding proteins recognise -The 5’ cap -The poly-A tail These then allow traffic through the nuclear pores Modulation of mRNA Transcription Many layers; 1. DNA binding -Enhancers -Repressors -Coactivators -Corepressors -Specific transcription factors (re:next slide) -Cytosine methylation 2. Histone modification -acetylation -deacetylation Modulation of mRNA Transcription by Cell Signals Ligand-receptor Nuclear hormone receptors Ligand second messenger systems Desequestration of Transcription factors Your Learning Objectives Your learning from today should focus on being able to; • Define transcription as the key event in gene expression during which the genetic code is read and transcribed into a portable template set of instructions for protein synthesis • Detail the sequence of events occurring during the initiation, elongation and termination of transcription • Describe the reactions catalysed by the spliceiosome and define what introns and exons • Provide examples of how and why the transcription rates of genes are often under exquisite regulation