1. Biological Modelling Gene Expression Data Neil Lawrence

2. Schedule Today: Introduction and Background 18 th AprilIntroduction and Background 25 th AprilcDNA Mircoarrays 2 nd MayNo Lecture 9 th MayAffymetrix GeneChips 16 th MayGuest Lecturer – Dr Pen Rashbass 23 rd MayAnalysis methods

3. Course Resources Website: http://www.dcs.shef.ac.uk/~neil. http://www.dcs.shef.ac.uk/~neil These slides Molecular Biology of The Cell –Alberts et al. http://www.thelifewire.com.http://www.thelifewire.com

4. Background DNA encodes all the manufacturing instructions for life. How are these instructions encoded? They are encoded in sequences – parallel course with Dr Gotoh.

5. The Genome All life forms have a genome. –Computers use binary sequences, biology uses quaternary sequences (base 4). Human Genome Project –Determines the sequences of genes in the human genome. –Leaves many unanswered questions – Human Proteome Project `Genes Were Easy’.

6. The Central Dogma DNA (as sequenced in HGP) makes RNA which makes protein – ‘The objects upon which life is based’ The process of DNA-RNA is known as transcription. Going from RNA-Protein is known as translation.

7. DNA Structure 1953 Crick and Wilkins, together with Watson, proposed the double-helix structure of DNA (Nobel Prize 1962). Sugar-phosphate backbone base pair

8. Coding in DNA We mentioned that sequences are quaternary. They are represented by chemical groups known as nucleotides. –T bonds with A and G bonds with C –sense and antisense Aadenine Gguanine Ccytosine Tthymine

9. RNA Coding Again in RNA, information is encoded in quaternary sequences. However now the nucleotide thymine is substituted with uracil. Aadenine Gguanine Ccytosine Uuracil

10. Proteins Proteins are sequences of amino acids. There are 20 amino acids (vigesimal sequences!). alaninecysteineaspartic acidglutamic acidphenylalanine glycinehistidineisoleucinelysineleucine methionineasparagineprolineglutaminearginine serinethreoninevalinetryptophantyrosine

11. Central Dogma Proteins are the functional units of life. What is the relation between proteins and DNA? The Central Dogma of Molecular Biology DNA makes RNA makes Protein

12. Prokaryotic Organisms Means no nuts in Greek – they have no nucleus. Example: E.Coli bacteria. We will use them to illustrate the Central Dogma.

13. RNA Polymerase Polymer-ase –An enzyme which makes polymers. RNA Polymerase copies DNA to an mRNA strand. It binds to a promoter region and finishes copying at a termination site. Try the transcription demo at: http://www.thelifewire.com/con_index.htm?12. http://www.thelifewire.com/con_index.htm?12

14. Transcription RNA Polymerase transcribes the DNA code to mRNA. The mRNA Polymerase `unzips’ the double helix of the DNA to access the code.

15. Transcription Schematic DNA Rewinding DNA double helix ribonucleotide triphosphates newly synthesized sense mRNA transcript for more accurate picture see pg 307, Alberts et al. antisense DNA strand 5’ 3’

16. Translation Nucleotides are grouped into codons. Each codon contains three nucleotides. Each codon corresponds to a protein or a stop. 2 nd Posi -tion 1 st Position3 rd Posi- tion UCAG U PheSerTyrCysU PheSerTyrCysC LeuSerSTOP A LeuSerSTOPTrpG C LeuProHisArgU LeuProHisArgC LeuProGlnArgA LeuProGlnArgG A IleThrAsnSerU IleThrAsnSerC IleThrLysArgA MetThrLysArgG G ValAlaAspGlyU ValAlaAspGlyC ValAlaGluGlyA ValAlaGluGlyG

17. tRNA and Ribosomes Transfer RNA (tRNA) translates the codons to amino acids. This takes place on a ribosome, which facilitates the building of the poly- peptide chain.

18. P SiteA Site E Site Amino Acids forming Peptide chain Ribosome tRNA anti-codon codon Translation UAC AUG Tyr GUA CAU Val mRNA strand 3’ 5’ HisMet Pro GGA CCU

19. Eukaryotic In Eukaryotic organisms transcription occurs in nucleus. –This protects it from the harsher environment of the cytoplasm. Translation occurs in cytoplasm.

20. Eukaryotic vs Prokaryotic Chromosome Chromosomes Eukaryotic Organism Prokaryotic Organism Transcription here Translation here

21. Gene Expression Now defined as the amount of mRNA produced from a particular gene. cf Protein Expression – the amount of protein produced from a particular gene. Regulated by many mechanisms –Not well understood! –Binding with anti-sense mRNA

22. Why Vary Protein Expression? Eukaryotic Cell differentiation (Haemoglobin only present in blood) Respond to environment (Glucocorticoids in the liver) Prokaryotic Respond to environment (see E.Coli example)

23. Example: E.coli Chromosome: contains 4.6 £ 10 6 nucleotide pairs, circular. DNA encodes approximately 4300 proteins. Only a fraction of these are made at any time. Expression is regulated according to available food.

24. Operon Model Five genes which manufacture an enzyme to produce the amino acid tryptophan are adjacent. Region starts with a promoter. promoterEDCBA operator operon When tryptophan is present in growth medium, a repressor protein binds to the operator repressing transcription. Activator proteins also exist.

25. Regulation in Eukaryotic Organisms Eukaroyotic organisms have more complex genetic switches. Regulation can be far more finely tuned. See Alberts et al. pg 396 and on.

26. Conclusions We have reviewed –The Central Dogma –Gene expression Coursework: Approx 1000 – 2000 word review of the mechanism of Gene Expression. Deadline Friday 10 th May