09BIS1012013TransciptLect9-1

The rnap was striped with phenol cleaned and

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Unformatted text preview: e 5' side of the lacZ gene of E. coli. lacZ E. Some of these mutations resulted in a decrease in lac mRNA and were lac therefore called "down promoter mutations." Our first impressions of Our the physical structure of the promoter came in 1975 when David Pribnow sequenced RNAP-protected DNA fragments to define the size and nucleotide sequence of the three bacteriophage T7 promoters. RNAP was bound to the T7 promoters and locked down with the dinucleotide 5’pppGpA. dinucleotide T7 DNA RNAP + dinucleotide 5’pppGpA BIS101­001, Spring 2013—Genes andGene Expression, R.L. Rodriguez ©2013 BIS101­001, Spring 2013—Genes and Gene Expression, R.L. Rodriguez 18 Promoter Structure: The Pribnow Experiment DNase was added to the T7 DNA and small DNA fragments (RNAP DNase protected DNA) were produced. The RNAP was striped with phenol, cleaned and sequenced. cleaned Pribnow identified three conserved sequences with fairly fixed spacing; Pribnow (1) -35 region, (2) the -10 or Pribnow box; and (3) the +1, the initiation -35 -10 Pribnow and +1 the nucleotide nucleotide + DNase GTTACA -35 TATPuATG Pu -10 +1 BIS101­001, Spring 2013—Genes and Gene Expression, R.L. Rodriguez ©2013 BIS101­001, Spring 2013—Genes and Gene Expression, R.L. Rodriguez +18 19 The Consensus Promoter The Hundreds of prokaryotic promoters have since been sequenced and found to have Hundreds highly conserved regions that correspond to Pribnow's consensus +1, -10 and -35 sequences. This has led to the concept of a consensus promoter sequence (Fig. consensus 8-12). A consensus promoter sequence is a hypothetical sequence based on the most commonly occurring nucleotides at a particular position. BIS101­001, Spring 2013—Genes and Gene Expression, R.L. Rodriguez ©2013 BIS101­001, Spring 2013—Genes and Gene Expression, R.L. Rodriguez 20 Consensus Promoters and Mutations In the sequence shown below, upper and lower case letters repr...
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