Transcription: Coupling Genotype to Phenotype - Biochemistry | BIOC 441, Study notes of Biochemistry

Download Transcription: Coupling Genotype to Phenotype - Biochemistry | BIOC 441 and more Study notes Biochemistry in PDF only on Docsity! 1 Exam Monday covers lectures 1-10. Remember: “Examinations are formidable even to the best prepared, for the greatest fool may ask more than the wisest man can answer.” Charles Caleb Colton-British clergyman and writer, ?1780-1832 5 Transcription What is a gene? How do we identify genes in biochemical/molecular terms? Three common ways (none foolproof): 1. DNA sequence >open reading frames (ORFs) (but introns…; genes encoding rRNA, tRNA, siRNA, snRNA). Genetic analysis: mutations. 2. RNA analysis >experimental:transcribed region of the gene (hybridization to an oligonucleotide array is best). 3. Binding analysis >in vitro and in vivo binding assays>regulatory regions Where does the RNA start? Where does it end? Which strand is transcribed? What DNA sequences are required to make the RNA-that is where are the signals for transcription to start and stop? 5’ 3’ 3’ 5’ DNA non-template (coding or sense) strand top strand DNA template (non-coding or anti-sense) strand bottom strand Genetically a gene is defined as a region of DNA that controls a discrete hereditary characteristic. The complete gene contains protein coding (exon) and non-coding (intron) DNA that is transcribed, as well as non-transcribed regulatory DNA. =transcribed region 6 Mapping the 5’ end of RNA and determining which strand is transcribed 5-P* Δ to denature Anneal 65oC with RNA RNA 5-P* 5-P* Δ to denature Sequence DNA 5-P* 5-P* Restriction enzyme digestion. Purify fragments 3’-end of DNA sequence corresponds to 5’ end of RNA 5’ 1. S1 nuclease mapping 2. Primer extension 3’ Extend with Reverse Transcriptase (copies RNA template into DNA) to sequence products Anneal with oligo Primer ( ) 3’ 5’ RNA preparation 5’3. DNA microarrays-how could you use DNA microarrays to map the 5’ end of RNA and determine which strand is transcribed? DNA 7 Measuring RNA levels in cells http://pathmicro.med.sc.edu/pcr/realtime-home.htmtt :// at icr . e .sc.e / cr/realti e e. t Isolate total RNA from cells Copy with reverse transcriptase (RT) using short (9 nucleotide), random (NNNNNNNNN) primers ( ) “real-time” PCR using a pair of primers( ) for the gene being assayed Problem: there are ~30,000 different RNAs, each present in a different amount in each human cell. Need a technique that is both very sensitive and very specific to detect and measure each RNA. Quantitative reverse transcriptase “real-time” PCR is the best method. PCR in the presence of a dye that fluoresces when it is bound to DNA measures product accumulation as a function of time. You can see from the graphs that this method is sensitive and quantitative over a 10e8-fold range!Plotting amount of DNA on a linear or a log scale 10 Structure of RNA polymerase from Thermus aquaticus Transcription process Transcription bubble DNA 3! | Nontemplate | strand nwinding Template strand dNTP channel RNA-DNA . hybrid, Active ~8 bp site Direction of transcription i 12 RNA polymerases: subunit composition Types of RNA polymerase enzymes: Size T7 bacterial virus:single polypeptide chain. 99kD Bacterial : five polypeptide chains. 450kD Eukaryotic: three nuclear enzymes, each containing 10-12 polypeptide chains ~600kD 15 Determining binding site for proteins on DNA 1. DNase “footprinting” 32P + * protein +DNase (*) * * * * * 16 DNase footprinting (cont.) Remove protein, run sequencing gel * * * * * * * * * * ** * “foot-print” { } Protected from digestion (-) (+) 17 Promoters Promoters are specific DNA sequences that determine where and how often transcription initiation occurs Bacterial promoter consensus sequence: -35 -10 +1 …ATG met RNA 20 Sigma and specific initiation 1. RNA pol in E. coli crude extracts will transcribe both double-stranded phage DNA and single-stranded DNA. 2. After passing the crude extract over a strong ion exchange column, activity on single-stranded DNA is recovered in the fraction containing α2, β, and β’ but no σ. These fractions had no activity on phage DNA which had no nicks or gaps. 3. Add-back experiments showed core polymerase in the active fractions and sigma was discovered in a fraction which would restore activity on the phage DNA. 4. Inference: sigma was required for transcription from genuine promoters; core would suffice for initiation if the DNA was single-stranded or contained nicks or gaps that created easily melted regions. 21 Elongation: RNA is synthesized from 5’ to 3’, copying DNA in a 3’-to-5’ direction 5’ 3’ The template DNA strand is also called the antisense strand 5’ end3’ end “Nascent RNA strand” N1’ N2 … Nx’ Nx+1’ The catalytic mechanism is essentially the same mechanism used by DNA polymerase, and all other nucleic acid polymerizing enzymes: an attack by the 3’OH at the end of the growing RNA chain on the α phosphate of the incoming NTP. 22 Transcription termination An RNA hairpin followed by a U- rich sequence causes termination of transcription and release of the RNA from the template and release of the enzyme from DNA.