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

2. Copyright-Anna Kramvis2 Central dogma of molecular biology http://www.cem.msu.edu/~reusch/VirtualText/nucacids.htm

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

4. 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)

5. 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

6. Prokaryotic genes Promoter region -35-10 Regulatory regions Open reading frame Initiation site Ribosome binding site Termination site

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

8. 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

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

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

11. Transcription and translation steps Prokaryotes Eukaryotes

12. Why the RNA step? MASTER COPY FLEXIBLE & DISPENSABLE

13. Transcription FINDING PROMOTER INITIATION ELONGATION

14. Transcription initiation

15. 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

16. Prokaryotic RNA Polymerase SIGMA FACTOR IS IMPORTANT FOR PROMOTER FINDING AND BINDING BY RNA POLYMERASE

17. Eukaryotic RNA Polymerase II

18. Transcription initiation from RNA Polymerase II

19. Regulation of transcription -activation PROMOTER RNA POLYMERASE  /TF ENHANCER CO-ACTIVATOR ADDITIONAL TRANSCRIPTION FACTORS ENHANCER PROTEIN

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

21. Sigma factors NH2COOH CORE BINDING REGION -10-35 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

22. 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

23. Regulation by sigma factors SPORULATION IN BACILLUS HEAT SHOCK RESPONSE

24. 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

25. 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.

26. 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

27. 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

28. 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

29. Antisense RNAs Complementary to a piece of mRNA Bind and prevent translation Important form of post-transcriptional regulation