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DNA RNA
Adenine Adenine
Cytosine Cytosine
Guanine Guanine
Thymine Uracil (U)
Tertiary structure
Classes of prokaryotic RNA
• ribosomal RNA (rRNA)
16S (small ribosomal subunit)
23S (large ribosomal subunit)
5S (large ribosomal subunit)
• transfer RNA (tRNA)
• messenger RNA (mRNA)
RNA polymerase
open promoter complex
initiation
elongation
termination
RNA product
Promoter structure in prokaryotes
mRNA
5’ PuPuPuPuPuPuPuPu AUG
-30 -10 +1
[ Promoter
]
transcription start site
mRNA
5’
-30 region -10 region
TTGACA TATAAT
AACTGT ATATTA
-36 -31 -12 -7 +1 +20
Pribnow box
T T G AC A TATA AT
82 84 79 64 53 45% 79 95 44 59 51 96%
consensus sequences
Prokaryotic RNA polymerase structure
2 uncertain
(Rifampicin target) 1 forms phosphodiester bonds
’ 1 binds DNA template
1 recognizes promoter and
facilitates initiation
’ ’ +
holoenzyme core polymerase sigma factor
The function of sigma factor
• the sigma subunit of RNA polymerase is an “initiation factor”
• there are several different sigma factors in E. coli that are
specific for different sets of genes
• sigma factor functions to ensure that RNA polymerase binds
stably to DNA only at promoters
• sigma destablizes nonspecific binding to non-promoter DNA
• sigma stabilizes specific binding to promoter DNA
• this accelerates the search for promoter DNA
Ka (M-1)
Any DNA Promoter DNA
(nonspecific) (specific)
Core 2 X 1011
A=T A = T
U=A U=A
• RNA synthesis usually initiated with ATP or GTP (the first nucleotide)
• RNA chains are synthesized in a 5’ to 3’ direction
The lactose operon in E. coli
• promoter binds CAP and RNA polymerase
• operator binds the lac repressor
promoter - operator
lac I P O lac Z lac Y lac A
-galactosidase
LACTOSE GLUCOSE + GALACTOSE
promoter - operator
lac I P O lac Z lac Y lac A
lac repressor
NO TRANSCRIPTION
NO TRANSCRIPTION
• IPTG (isopropyl thiogalactoside)
is also used as a (non-physiological) inducer
• repressor (with bound allolactose) dissociates from the operator
• negative control (repression) is alleviated, however...
NO TRANSCRIPTION
RNA pol
1
Specificity is the ratio of
(Ka for binding to operator DNA) / (Ka for binding to random DNA)
Regulation of the lactose operon - positive control
2
also termed catabolite activator protein NO TRANSCRIPTION
Activation of lac operon transcription
RNA pol
lac I lac Z lac Y lac A
inactive repressor