Gene Expression

1. Gene Expression 1: Activation (overview), Transcription, Translation.  The Goodwin Equations

2. Review • An active gene is one that is being transcribed, and whose transcription product is being translated, and whose translation product affects cellular behavior/development. • Genes are activated by transcription factors – the principal way transcription is controlled • Transcription factors are proteins that themselves are gene products. • Transcription factors often require a cofactor, a very important environmental signal.

3. Gene Activation is a control system Thus, one has a rudimentary feedback loop

4. Since the transcription factor is itself a gene product the feedback loop can (and usually does) involve multiple genes. Since the cofactor level may involve environmental stimuli the feedback loop can involve their signal transduction pathways.

5. Gene Expression is best understood by mapping, quantifying, and understandings the behavior of these loops.

6. Transcription • A good reference: (Blackwell, 2001)

7. Genes are transcribed (copied) into 'messenger RNA' by an enzyme called RNA Polymerase [RNAP]. • Polymerase binds to a 'promoter region' at the beginning of a gene. • The polymerase traverses the gene, copying as it goes. • Polymerase normally leaves DNA at the gene's end. • Multiple polymerases may be attached to one gene at any given time.

8. The transcription “bubble”, White, Fig. 1.8

9. Three kinds of RNAP • Pol I – rRNA • Pol II – mRNA and snRNA (small nuclear RNA, involved in splicing) • Pol III – small RNA’s (tRNA, 5s rRNA …) We will concentrate on Pol II although many features are common to all three.

10. Model of Pol II RNAP(White)

11. What determines the rate of transcription? • Transcription velocity is mostly constant, over one gene and from gene to gene. • Transcription length is determined by the gene. Thus … • (Molar) synthesis rate for transcription is controlled by gene length, number of RNAP's on the gene. • Rates (Hargrove): 2500 nt min-1 (procaryotes), 3000 nt min -1 (eukaryotes)

12. The initiation rate for transcription* is the most important factor determining which gene products are generated *i.e. the attachment and hence (in steady state) the detachment rate for RNA polymerase (RNAP). This determines the average number of mRNA's on the gene.

13. What determines the rate of translation (mRNA protein)? • Translation is effected by ribosomes, complex enzymes made of both protein and nucleic acid, that traverse mRNA's and translate their codons (RNA triplets) into amino acid sequences. • In a manner similar to transcription, the ribosome traverses at a near-constant velocity. Thus …

14. for translation … • (Molar) synthesis rate is controlled by ribosomal attachment rate, mRNA length, and the number of mRNA's present. • Rates (Hargrove): 2700 nt min-1 (procaryotes), 720 nt min -1 (eukaryotes)

15. Summary of Gene Expression Rates Typical values for parameters and rates needed to quantify gene expression in E. coli and mammalian cells (from Hargrove)

16. The train-on-the-track model Rate = Number of tracks x Number of trains x Velocity of trains / Track length

17. Critical Factors: • For transcription – the attachment rate, since the number of gene copies (one or two), transcription velocity and length are fixed. • For translation – the number of mRNA's present, since the ribosomal attachement rate, translation velocity and length are fixed.

18. Summary of Steps in Eukaryotic Processing

19. What is the RNAP “train starter”? • Transcription factors. • Inducers • Repressors • These are protein molecules, made by genes, that bind to a gene at an operator site, in or near a promoter region, upstream of where transcrip-tion takes place. They often exist in two forms quiescent and active. Usually a small molecule induces the change: Inactive factor  small molecule  active factor

20. Transcription FactorsIt is important to remember that transcription factors are proteins, come from genes (like all proteins), and may influence either their predecessor gene or –often– other genes. Summary of the structure of the Engrailed homeodomain bound to DNA, as revealed by X-ray crystallography. Cylinders represent the three -helices of the homeodomain, ribbons represent the sugar phosphate backbone of the DNA and bars symbolize the base pairs. The recognition helix (3) is shown in red.

21. Transcription factors have many shapes and thus modes of interaction with DNA

22. Promoters (pol II) • Contain multiple binding sites for transcription factors. • Other binding sites upward, downward of (enhancers), and within transcribed region.

23. Basal Transcription Factors • TATA box – TATAa/tAa/ t • Initiator – YYANa/ tYY, where Y is a pyrimidine, N is any base, and transcription begins at the A • Picture shows assembly of basal pol II on adenovirus ML promoter

24. Preassembly of the Complex is possible: kinetic implications?

25. Actual Initiation (White, p. 62) • Promoter melting – DNA strands separate. • Initiation – first RNA phosphodiester bond is formed. • Clearance – pol II released from the factors assembled at the promoter. Elongation and Termination • Elongation factors • Termination Sequence (AAUAAA) and cleavage factor. Polyadenylation.

26. Enhancers (White, p. 73) Enhancer region located ~ 1 kb upstream of mouse muscle creatine kinase gene. There are at least 6 different transcription factors with expression “governed by combinatorial interactions amongst the transcription factors”.

27. Transcription factor activation

28. Transcription Factor Production, one example. (White, p. 170)

29. Transcription Control by Stimulated Translocation

30. Transcription of the WT1 Gene Negative feedback: WT1 protein inhibits expression of its own gene and also that of PAX-2 an activator of th WT1 promoter.

31. Myogenesis Upstream regulators force differentiation to mesodermal precursor cells that then express bHLH proteins that stimulate transcription of their own genes. They also activate genes that make MEF2, which further accelerates transcription of genes for bHLH proteins. MEF2 and bHLH proteins both stimulate other muscle-specific genes. Positive feedback!

32. In general, transcription factors and the molecules that activate them are crucial to determining the array of genes that are on.

33. Transcription factors determine mRNA production; do they determine mRNA number?(mRNA number is what determines translation)

34. mRNA number is determined by a balance between generation and consumption rate: This is the first part of the Goodwin equation set.

35. mRNA numbers determine protein production; do they determine translation product levels?

36. Translation product levels are determined by a balance between their generation and consumption rates: This is the second part of the Goodwin equation set.

37. Frequently a translation product will mediate the level of another metabolite (galactosidase, tryp enzymes) • This can give rise to a third set of Goodwin equations. • Product levels determined by the third set often provide the "loop-closing" feedback.

38. What has been covered?