Transcription Nicky Mulder Acknowledgements: Anna Kramvis for lecture material (adapted here)

Copyright-Anna Kramvis2 Central dogma of molecular biology

DNA -> RNA -> PROTEIN GENES are transcribed/expressed Converts information from DNA into a usable template for protein

Copyright-Anna Kramvis4 What is a gene? Segment of DNA that is transcribed into RNA This RNA can be:  Non-coding –rRNA, tRNA, siRNA, antisense RNA  Protein coding –messenger RNA (mRNA)

Copyright-Anna Kramvis5 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

Prokaryotic genes Promoter region Regulatory regions Open reading frame Initiation site Ribosome binding site Termination site

Prokaryotic genes cont. Can be encoded on different strands: forward or reverse Promoter Some are in operons One transcript

Eukaryotic genes Many eukaryotic genes include introns and exons Coding part is in the exons which need to be joined 3’ exonexonintron open reading frame upstreamdownstream 5’ initiation codon termination codon

Eukaryotic gene reality Small exons, large introns! Exons are joined by splicing – can lead to multiple products

Alternative splicing Exon 1Intron 1Exon 3Exon 2Intron 2 Exon 1Exon 2Exon 3 Exon 1Exon 2 Splice junction

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 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 PROMOTER RNA POLYMERASE  /TF ENHANCER CO-ACTIVATOR ADDITIONAL TRANSCRIPTION FACTORS ENHANCER PROTEIN

Regulation of transcription -repression PROMOTER RNA POLYMERASE  /TF ENHANCER ANTI-  FACTOR REPRESSOR RBS - HISTONE DEACETYLATION -DNA METHYLATION -CHROMATIN STRUCTURE -AUTOREGULATION

Sigma factors NH2COOH CORE BINDING REGION Transcription factors have DNA binding region and protein interaction domain Sigma factor structure  RNA POLYMERASE MAJOR  FACTORS - HOUSEKEEPING GENES  RNA POLYMERASE   STRESS RESPONSE EXTRACYTOPLASMIC OTHER FUNCTIONS ALTERNATIVE  FACTORS

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 NF-KB TP1 AP-1 STAT Multicellular organisms need intra-cellular signals controlled by gene expression. Up to 10% of human genes may encode TFs.

Transcription regulation and human disease Heart failure - Switch between 2 different metabolic pathways Huntington’s disease - polyglutamine stretches & transcription dysregulation Cancer - oncogenes and anti- oncogenes 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