4. RNA synthesis occurs in a moving transcription bubble on the DNA template o Only a short RNA-Dna hybrid(8 bp in bacteria) is present through the transcription process At each moment, a region of about l7 bp on the e coli dna is unwound in the transcription bubble The rna chain is extended at a rate of 50-90 nucleotides/ second by the E coli rna polymerase o Unwinding ahead of and rewinding behind of the transcription bubble produces positive and negative supercoils respectively on the dna (relieved by the action of topoisomerases)
4. RNA synthesis occurs in a “moving” transcription bubble on the DNA template ⚫ Only a short RNA-DNA hybrid (~8 bp in bacteria) is present through the transcription process. ⚫ At each moment, a region of about 17 bp on the E. coli DNA is unwound in the transcription bubble. ⚫ The RNA chain is extended at a rate of 50-90 nucleotides/second by the E. coli RNA polymerase. ⚫ Unwinding ahead of and rewinding behind of the transcription bubble produces positive and negative supercoils respectively on the DNA (relieved by the action of topoisomerases)
Transcription bubble Nontemplate strand RNA polymerase Rewinding Unwinding DNA 3 3 stran d RNA 6′RNA-DNA Active site hybrid, 8 bp Direction of transcription (5)CGCTATAGCGTTT(3) DNA nontemplate(coding)strand (3)GCGATATCGCAAA(5) DNA template strand (5) CGCUAUAGCGUUU(3) RNA transcript
Negative supercoils Positive supercoilS 5 RNA Direction of transcription (b)
5. RNA polymerase recognizes specific promoter sequences on DNA to initiate transcription o Promoter sequences are located adjacent to genes o Promoters can be identified using" protection assays (e.g, footprinting techniques) o Promoters, although all bind to the same polymerase have quite variable DNa sequences(surprisingly), but with two consensus sequences centered at-10 and 35 positions (the first residue of the rna is given +1) o Promoters having sequences more similar to the consensus are more efficient, and vice versa (from studies of mutations and activity comparison)
5. RNA polymerase recognizes specific promoter sequences on DNA to initiate transcription ⚫ Promoter sequences are located adjacent to genes. ⚫ Promoters can be identified using “protection assays” (e.g., footprinting techniques). ⚫ Promoters, although all bind to the same polymerase, have quite variable DNA sequences (surprisingly), but with two consensus sequences centered at –10 and – 35 positions (the first residue of the RNA is given +1). ⚫ Promoters having sequences more similar to the consensus are more efficient, and vice versa (from studies of mutations and activity comparison)
The footprinting P end-labeled DNA technique Specific binding protein protein DNase nicks DNase nicks randoml !!!!!! ↓⌒↓↓↓ The footprint 32P-labeled Gel electrophoresis 32P-labeled fragments pattern fragments
The footprinting technique The footprint - protein + protein randomly