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Transcription Nicky Mulder Acknowledgements: Anna Kramvis for lecture material (adapted here) Central dogma of molecular biology http://www.cem.msu.edu/~reusch/VirtualText/nucacids.htm Copyright-Anna Kramvis 2 DNA -> RNA -> PROTEIN GENES are transcribed/expressed Converts information from DNA into a usable template for protein What is a gene? Segment of DNA that is transcribed into RNA This RNA can be: –rRNA, tRNA, siRNA, antisense RNA Protein coding –messenger RNA (mRNA) Non-coding Copyright-Anna Kramvis 4 Parts of genes The part of a coding gene that is translated into a protein is the Open Reading Frame (ORF) Within the ORF, triplets of bases (codons) code for amino acids via the genetic code An ORF starts with an initiation codon and ends with a stop codon Copyright-Anna Kramvis 5 Prokaryotic genes Promoter region -35 Open reading frame -10 Initiation site Regulatory regions Ribosome binding site Termination site Prokaryotic genes cont. Can be encoded on different strands: forward or reverse Some are in operons Promoter One transcript Eukaryotic genes Many eukaryotic genes include introns and exons Coding part is in the exons which need to be joined open reading frame 5’ upstream exon initiation codon intron exon downstream termination codon 3’ Eukaryotic gene reality Small exons, large introns! Exons are joined by splicing – can lead to multiple products Alternative splicing Splice junction Exon 1 Exon 2 Intron 1 Exon 2 Exon 1 Exon 2 Exon 3 Exon 1 Intron 2 Exon 3 Exon 3 Exon 2 Exon 3 Transcription and translation steps Prokaryotes Eukaryotes Why the RNA step? MASTER COPY FLEXIBLE & DISPENSABLE Transcription FINDING PROMOTER INITIATION ELONGATION Transcription initiation RNA Polymerase enzymes Prokaryotes: 1 RNA polymerase, 4 subunits , , ’, Eukaryotes: 3 different RNA polymerases, I, II, III each 12-16 subunits II is most well studied Prokaryotic RNA Polymerase SIGMA FACTOR IS IMPORTANT FOR PROMOTER FINDING AND BINDING BY RNA POLYMERASE Eukaryotic RNA Polymerase II Transcription initiation from RNA Polymerase II Regulation of transcription -activation ADDITIONAL TRANSCRIPTION FACTORS /TF ENHANCER ENHANCER PROTEIN RNA POLYMERASE PROMOTER CO-ACTIVATOR Regulation of transcription -repression ANTI- FACTOR /TF ENHANCER RBS RNA POLYMERASE REPRESSOR REPRESSOR REPRESSOR PROMOTER -HISTONE DEACETYLATION -DNA METHYLATION -CHROMATIN STRUCTURE -AUTOREGULATION Sigma factors Transcription factors have DNA binding region and protein interaction domain Sigma factor structure -10 -35 NH2 COOH CORE BINDING REGION MAJOR FACTORS HOUSEKEEPING GENES RNA POLYMERASE ALTERNATIVE FACTORS RNA POLYMERASE STRESS RESPONSE OTHER FUNCTIONS EXTRACYTOPLASMIC Regulation of factors Autoregulation of its own expression Regulation of expression by external signals Half-life of RNA or protein Relative abundance of different factors Regulation by anti-sigma factors Proximity of the sigma factors Regulation by sigma factors SPORULATION IN BACILLUS HEAT SHOCK RESPONSE Transcription and drug targets RNA polymerase subunit is target for anti-TB drug rifampicin SigB regulates katG- involved in INH resistance Other transcription factors that have a core role in the control of expression of specific sets of genes eg IdeR Gene regulation in humans Multicellular organisms need intra-cellular signals controlled by gene expression. Up to 10% of human genes may encode TFs. NF-KB TP1 AP-1 STAT Transcription regulation and human disease Heart failure - Switch between 2 different metabolic pathways Huntington’s disease - polyglutamine stretches & transcription dysregulation Cancer - oncogenes and antioncogenes overexpression of genes, P53 Gene expression is NB Having the right proteins at the right time No waste in producing what you don’t need Being able to respond quickly to changes Getting the right regulators present to control gene expression Additional features of RNAs Have ability to fold into secondary structures RNAs can be catalytic –ribozymes RNAs can regulate transcription or translation RNAs can be genomes Antisense RNAs Complementary to a piece of mRNA Bind and prevent translation Important form of post-transcriptional regulation
