lecture 15- transcription in bacteria Flashcards
describe bacterial RNA polymerase core
5 subunits: 2 alpha, 1 beta, 1 beta’, 1 w
- look like crab claws
- capable of RNA synthesis on a DNA template
- however… no specificity for promoter & no initiation in vivo
describe bacterial RNA polymerase holoenzyme
6 subunits:
the core (2 alpha, 1 beta, 1 beta’, 1 w) + sigma factor
- core required for polymerization activity
- sigma factor required for correct initiation of transcription- binding to promoter
the sigma factor directs the RNA polymerase holoenzyme to ___
specific binding sites on the DNA
describe the different sigma factors in E. coli
E. coli has several sigma factors that specify RNA polymerase binding to particular promoters
- because diff sigma factors direct to diff promoters, different sets of genes may be transcribed as needed by changing the sigma factor in the holoenzyme
most common sigma factor in E. coli
sigma 70- used for housekeeping genes expressed in all growing cells
-35: TTGACA
-10: TATAAT
16-18 bp
what is a consensus sequence
although sequences not identical for all bacterial promoters, certain nucleotides that are particularly common form consensus sequence - determined by aligning all known examples and finding most common base at each position
consensus sequence of sigma 70 promoter: sequence of most of the promoter is ___, only short stretches of DNA are ___
irrelevant
conserved
describe features of E. coli promoters recognized by sigma 70
consensus sequences (interaction sites for sigma 70 are -10 and -35
-10: 5’-TATAAT-3’
-35: 5’-TTGACA-3’
17 bp distance between the two
- upstream promoter element = promoters of certain highly expressed genes (bound by 1 alpha subunit of RNA polymerase)
what do mutations in the -10 and -35 regions of the promoter do?
affect efficiency of RNA polymerase binding and transcription initiation
- a change in just one base pair can decrease rate of binding by several orders of magnitude
describe how structural changes lead to formation of the transcription- competent open complex
transition to open complex and elongation requires conformational changes in RNA polymerase and changes of its association with DNA
- place downstream duplex DNA in the active site cleft and separate nontemplate and template strands (DNA melting- open complex)
- open complex through spontaneous isomerization
how does RNA polymerase allow access of the DNA to the core of the enzyme?
several distinct channels in RNA polymerase allow DNA to enter as dsDNA and to peel apart within the polymerase so that 8bp form between the template strand and the growing RNA transcript
- 2 other channels provide entry for rNTP’s and an exit for the transcript
initiation of transcription is ___-independent and produces ___
describe this
primer
short, abortive transcripts
- the first 8-10 phosphodiester bonds formed: high probability that RNA polymerase will release transcript from template without extending it further
- beyond 10 nuc’s: RNA becomes stable, release of sigma factor
what is promoter clearance
RNA polymerase moves beyond promoter region of DNA to begin rapid elongation of the transcript
two types of terminators in E. coli
1- intrinsic (rho-independent) terminators- relies primarily on structures that form in the RNA transcript
2- Rho-dependent- require rho protein
what are the two distinguishing features of rho-independent terminators
1- highly conserved segment of adenine residues in template strand that are transcribed into uracil residues
2- RNA transcript with self-complementary sequences - formation of hairpin structure centered 15-20 nuc’s before projected end of RNA strand
