Slide 1Genomic DNA sequence as an algorithmSlide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 421Bi 1 “Drugs and the Brain” Lecture 17 Tuesday, May 2, 2006From the Genome to mRNA21. 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.32. Complete DNA sequence as scripturebasic sequenceRNA sequenceprotein sequenceprotein structureRNA splicingmutations that cause diseasesingle-nucleotide polymorphisms (SNPs)orthologs in other speciesprotein functionproteins that bind to the sequence and regulate expression chromosomal locationRNA abundance4http://www.ncbi.nih.gov/Database/datamodel/index.html2. Complete DNA sequence as scripture522,000 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 regions3. Introns We now describe the rest6Lander Figure 171. Repetitive elementsEncode proteins7Little Alberts Fig. 8-15© Garland publishing2. Gene activation involves regulatory regions from Lecture 148Gene (DNA)proteincoding sequencesnoncoding sequences3. Introns and Exonsmessenger RNA (mRNA)translated sequencesuntranslated sequencesexon introntranslationsplicing (introns removed)transcription (mRNA synthesis)9Exons don’t differ much among organisms, Lander et al Figure 35but human introns are longer10Humans 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 DNA2. Human genes are used to construct more alternative transcripts. Result: humans have ~ 5 times as many protein products as worms or flies.11From past lectures, some pictures of RNA polymeraseorTranscription factors12in vitro RNA synthesisRNA polymerase promoterDNAmeasureSite-Directed Mutagenesis on Ion ChannelsExpress by injecting into immature frog eggsMutate the desired codon(s)measure(from Lecture 7)13RNA polymerase promoterRNA polymeraseDNAStep1:bind “nonspecifically” to DNAStep 2:bind “specifically” to promoter(from Lecture 10)One-dimensional diffusion: a protein bound to DNA14Many genes have a DNA sequence called“cAMP-Ca2+ responsive element”(CRE)The protein that binds to this CRE is called“cAMP-Ca2+ responsive element binder”(CREB). CREB binds in its phosphorylated form, called pCREB.pCREB is a “transcription factor”.-O OPOOkinasephosphorylatedproteincAMPCa2+intracellularmessenger(from Lecture 14)15Directed Mutagenesis Applied to Control Elements in DNA(from Lecture 14)16(from Lecture 14)andNestlerFigure 16-517Proteins bind to specific but limited stretches of DNA(8 - 20 base pairs)kinasephosphorylatedproteincAMPCa2+intracellularmessengerhttp://www.its.caltech.edu/~lester/Bi-1/Transcription factor-DNA complex.pdb(Swiss-prot viewer must be installed on your computer)(from Lecture 14)18Little Alberts Fig. 8-15© Garland publishingfrom Lecture 1419Viewing a single DNA-protein complex, #2Atomic Force MicroscopeSampleCantilever with tipSegmented photodiodeLaser(#1 was Steve Quake’s single-molecule DNA sequencing experiment, Lecture 16)20Tip and cantileverof anatomic force microscope21Single-molecule AFM image of two protein molecules bound to DNAkinasephosphorylatedproteincAMPCa2+intracellularmessenger22Little Alberts Fig 7-7© Garland publishing23To build RNA (or DNA)ribonucleic acid (or deoxyribonucleic acid),the cell begins with nucleotides, for instance ATPhydrogen bonds to U(to T in DNA)The BaseAU (T in DNA)CGThe phosphates4 negative charges;2 are usually neutralized by Mg2+ The 5-carbon sugarribose(2’-deoxyribose in DNA)3’ 2’5’NNNNNH2OOHOHHHCH2HOPO-OOPO-O-O OPO-OMg2+deoxyDNAH24OHLittle Alberts Fig 3-42© Garland publishingligatenickLatin, to tie25DNA is quite stableA mosquito with a parasitic mite, caught in amber ~ 25 mybutRNA is quiteunstable26How fast does the RNA chain grow? Macroscopic measurementsA. Outside the lab:1. Build nuclear reactor2. Irradiate [32S]sulfate to produce [32P]phosphate 3. Phosphorylate adenosine with [32P]phosphate, yielding [32P]-AMP. 4. Phosphorylate twice more to produce -[32P]ATP27B. Inside the lab:5. Order -[32P]ATP6. Add -[32P]ATP to reaction 7. Separate mononucleotides from polymers (RNA)8. Count radioactive decays in polymers9. Result: elongation varies with nucleotide concentration, but the maximal rate is ~ 30 nt/s.How fast does the RNA chain grow? Macroscopic measurements281. 32P half-life 14 d. Time constant = t/ln(2) = 20.6 d, rate constant = 0.048/d = 3.4 x 10-5 /min 2. If we would like 100 dpm (disintegrations per min), we need (100 dpm)/(3.4 x 10-5 /min) = 3.0 x 106 atoms of 32P. 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 106 / 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?291mDNA (double-stranded) How many nucleotides (nt)? 1.5 m = 1.5 x 104 Å.(1.5 x 104 Å)/ (3.4 Å/ nt) = 4400 ntRNA 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 7-830Where does the energy go? Dynamic single-molecule measurementsE = force x distance;force is generated by viscosityRNA polymerase31Movie of the RNA polymerase experimentsDescription 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