1 Bi 1 “Drugs and the Brain” Lecture 17 Tuesday, May 2, 2006 From the Genome to mRNA

2 1.Genomic DNA sequence as an algorithm 1. The genome contains the “parts list” PLUS the rules for parts use. 2. The major rules for “parts use” operate through differential gene expression. Now, the goals: to understand both -the complete parts list and -the rules for use.

3 2. Complete DNA sequence as scripture basic sequence RNA sequence protein sequence protein structure RNA splicing mutations that cause disease single-nucleotide polymorphisms (SNPs) orthologs in other species protein function proteins that bind to the sequence and regulate expression chromosomal location RNA abundance

Complete DNA sequence as scripture

5 22,000 genes x 400 codons/protein x 3 bases/codon = 26.4 million base pairs, or < 1% of the genome! How much coding sequence is in the genome? 1.Repetitive elements (junk? selfish DNA?) 2.Regulatory regions 3.Introns We now describe the rest

6 Lander Figure Repetitive elements Encode proteins

7 Little Alberts Fig © Garland publishing 2. Gene activation involves regulatory regions from Lecture 14

8 Gene (DNA) protein coding sequences noncoding sequences 3. Introns and Exons messenger RNA (mRNA) translated sequences untranslated sequences exonintron translation splicing (introns removed) transcription (mRNA synthesis)

9 Exons don’t differ much among organisms, Lander et al Figure 35 but human introns are longer

10 Humans have less than twice as many genes as worm or fly. However, human genes differ in two ways from those in worm or fly. 1.Human genes are spread out over much larger regions of genomic DNA 2.Human genes are used to construct more alternative transcripts. Result: humans have ~ 5 times as many protein products as worms or flies.

11 From past lectures, some pictures of RNA polymerase or Transcription factors

12 in vitro RNA synthesis RNA polymerase promoter DNA measure Site-Directed Mutagenesis on Ion Channels Express by injecting into immature frog eggs Mutate the desired codon(s) measure (from Lecture 7)

13 RNA polymerase promoter RNA polymerase DNA Step1: bind “nonspecifically” to DNA Step 2: bind “specifically” to promoter (from Lecture 10) One-dimensional diffusion: a protein bound to DNA

14 Many genes have a DNA sequence called “cAMP-Ca 2+ responsive element” (CRE) The protein that binds to this CRE is called “cAMP-Ca 2+ responsive element binder” (CREB). CREB binds in its phosphorylated form, called pCREB. pCREB is a “transcription factor”. kinase phosphorylated protein cAMP Ca 2+ intracellular messenger (from Lecture 14)

15 Directed Mutagenesis Applied to Control Elements in DNA (from Lecture 14)

16 (from Lecture 14) and Nestler Figure 16-5

17 Proteins bind to specific but limited stretches of DNA ( base pairs) kinase phosphorylated protein cAMP Ca 2+ intracellular messenger factor-DNA complex.pdb (Swiss-prot viewer must be installed on your computer) (from Lecture 14)

18 Little Alberts Fig © Garland publishing from Lecture 14

19 Viewing a single DNA-protein complex, #2 Atomic Force Microscope Sample Cantilever with tip Segmented photodiode Laser (#1 was Steve Quake’s single-molecule DNA sequencing experiment, Lecture 16)

20 Tip and cantilever of an atomic force microscope

21 Single-molecule AFM image of two protein molecules bound to DNA kinase phosphorylated protein cAMP Ca 2+ intracellular messenger

22 Little Alberts Fig 7-7 © Garland publishing

23 To build RNA (or DNA) ribonucleic acid (or deoxyribonucleic acid), the cell begins with nucleotides, for instance ATP hydrogen bonds to U (to T in DNA) The Base A U (T in DNA) C G  The phosphates 4 negative charges; 2 are usually neutralized by Mg 2+ The 5-carbon sugar ribose (2’-deoxyribose in DNA) 3’ 2’ 5’ deoxy DNA H

24 OH       Little Alberts Fig 3-42 © Garland publishing ligate nick Latin, to tie

25 DNA is quite stable A mosquito with a parasitic mite, caught in amber ~ 25 my but RNA is quite unstable

26 How fast does the RNA chain grow? Macroscopic measurements A. Outside the lab: 1. Build nuclear reactor 2. Irradiate [ 32 S]sulfate to produce [ 32 P]phosphate 3. Phosphorylate adenosine with [ 32 P]phosphate, yielding [ 32 P]-AMP. 4. Phosphorylate twice more to produce  -[ 32 P]ATP

27 B. Inside the lab: 5. Order  -[ 32 P]ATP 6. Add  -[ 32 P]ATP to reaction 7. Separate mononucleotides from polymers (RNA) 8. Count radioactive decays in polymers 9. Result: elongation varies with nucleotide concentration, but the maximal rate is ~ 30 nt/s. How fast does the RNA chain grow? Macroscopic measurements

P half-life 14 d. Time constant = t/ln(2) = 20.6 d, rate constant = 0.048/d = 3.4 x /min 2. If we would like 100 dpm (disintegrations per min), we need (100 dpm)/(3.4 x /min) = 3.0 x 10 6 atoms of 32 P. 3. Assume that we have an RNA molecule encoding a protein of length 330 amino acids, or 1000 nucleotides. On the average, 25% of these will be ATP. Therefore we need a number of RNA molecules equal to 3.0 x 10 6 / 250, or 11,900 RNA molecules. 4. The least abundant mRNAs occur at abundances of one/cell; the most abundant, ~ 10,000/cell. Therefore we need between 1 and 12,000 cells. How much material do we need for macroscopic measurements?

29 1  m DNA (double-stranded) How many nucleotides (nt)? 1.5  m = 1.5 x 10 4 Å. (1.5 x 10 4 Å)/ (3.4 Å/ nt) = 4400 nt RNA being synthesized (single-stranded) Electron micrograph of RNA synthesis from a DNA template ~ 110 molecules of RNA polymerase (not visible) “latest” ~ 500 s “earliest” Little Alberts Fig 7-8

30 Where does the energy go? Dynamic single-molecule measurements E = force x distance; force is generated by viscosity RNA polymerase

31 Movie of the RNA polymerase experiments Description of the movie Approximately one frame every 5 seconds was selected to speed up the movement of the transcription process. A total of ~10 minutes of transcription is displayed and the movie is "looped" 8 times. When a new loop starts the total shortening of the DNA tether is clearly visible. (your choice of several formats)

32 RNA polymerase travels at a “constant” rate until it stalls stall at 30% - 60% efficiency

33 DNA has well-understood chemistry and is stable RNA has well-understood chemistry but is unstable Proteins have complex chemistry and a wide range of stability Constraints on a “complete” or systems approach to biology

34 1.Make a DNA array from the genome of interest 2.Extract mRNA (wear those rubber gloves!) from the organ of interest. (Control vs experiment) 3.Reverse transcribe / label with fluorescent dyes (make “complementary DNA” or cDNA) 4.Hybridize to array (use those base-pairing rules) 5.Read with scanner Steps in Microarray Analysis

35 1. One way to generate a DNA microarray: photochemistry Unreactive, but photosensitive moiety spot1 spot2 spot3 spot4 spot5

36 1. Another way to generate a DNA microarray: PCR + pens From Lecture 15: Thousands of primer pairs Collection of standard 96- well dishes Many identical slides, each with thousands of spots 7

37 Little Alberts Figure , 3. Generating cDNA with reverse transcriptase

38 4. Next slide: An experiment to determine different gene expression in schizophrenic vs control brains From Dr. David Lewis Visiting psychiatry lecture Bi Most striking result: a decreased level of mRNA for an auxiliary protein in the G protein cycle

nm CONTROLSCHIZOPHRENIA Isolate total RNA Isolate mRNA Reverse transcibe (make cDNA) Cy3Cy5 Combine and Hybridize Harvest Area 9 UniGEM-V ~7,000 genes Combined Image Fulton & Weinberger, 1999 AAAA AAAAA AAAA AAAAA * * * ** * * * ** AAAA AAAAA AAAA UniGEM-V ~7,000 genes

40 5. Microarray scanners are now about as large as a microwave oven or boom box or desktop PC. For example, The software for analyzing 10 4 to 10 5 spots per slide is quite advanced Emphasizing the limitations of mRNA  cDNA microarrays: Translation rates, RNA stability, protein stability

41 messenger RNA (mRNA) Gene (DNA) protein coding sequences noncoding sequences Components of Expression translated sequences untranslated sequences exonintron translation (Lecture 18) splicing (introns removed) transcription (mRNA synthesis)

42 Bi 1 “Drugs and the Brain” End of Lecture 17