Promoter sequence defines two key decisions

+1 3’
RNA AGCU
(nontemplate strand)
5’ AGCT coding strand
DNA
3’ TCGA template strand

promoter terminator

Which DNA strand serves as the template

Where does transcription start

1
221 S16
 Prokaryotic transcription: initiation

2
221 S16
 E. coli RNA polymerase

subunit stochiometry
protein core holoenzyme elongating
Gene function
subunit polymerase initiation competent termination competent

promoter
rpoA a recognition; 2 2 2
bind activators

rpoB b catalytic 1 1 1

rpoC b’ catalytic 1 1 1
assembly of s
rpoZ  factors 1 1 1
promoter
rpoD s70 specificity 1

aabb’  aabb’ s70) aabb’

3
221 S16
 E. coli RNA polymerase

cis-acting element
A site on a DNA (or RNA) molecule that
functions as a regulatory site – often a
binding site for a sequence-specific binding
protei.
e.g., promoter

trans-acting factor
A diffusible regulatory molecule - often a
protein - that binds to a specific cis-acting
element

e.g., RNA polymerase

The trans-acting factor binding to its cognate cis-acting element affects only
adjacent DNA (or RNA) sequences on the same molecule.
4
221 S16
 Typical prokaryotic promoter

RNAP binding at -10 and -35 region

Local unwinding/melting at -10 region

5
221 S16
 E. coli RNA polymerase – DNA interactions during initiation

6
221 S16 Jie Chen et al. PNAS 2010;107:12523-12528
 E. coli RNA polymerase – DNA interactions during initiation

template strand

sigma70
protein domains

closed complex open complex

7
221 S16 Chen et al. PNAS 2010;107:12523-12528
 E. coli promoter alignment

Pribnow box

consensus sequence
a generalized base sequence
derived from closely related sequences
where each position in the consensus sequence
is found in the majority of sequences at that position
8
221 S16
 E. coli promoter alignment

Sequence logo

-35 -10
Sequence logos are a graphical technique
for displaying a summary of a set of aligned 263 strong promoters
sequences.
9
221 S16 Dekhtyar et al. (2008) BMC Bioinformatics 2008, 9:233
 E. coli RNA polymerase: promoter binding

(sigma70 domains)
Biochemical evidence
Physical evidence

17 bp

Genetic evidence

usually affects usually affects

RNAP binding RNAP binding
also can affect
template melting 10
221 S16
 E. coli RNA polymerase binds to
one side of the double-helix

-35

-10

17 bp

11
221 S16
 E. coli RNA polymerase binds to one side of the double-helix

consensus:
-35 -10

+2 bp
insertion
mutation:

-37 -10

12
221 S16 221S’14
 Prokaryotic transcription: factor-independent termination

loop
Two types of termination:
• rho-independent (intrinsic)
• rho-dependent
Intramolecular “stem-loop”
G:C-rich stem
U-rich 3’-end

13
221 S16
 Prokaryotic transcription: factor-independent termination

Palindrome is
A word, phrase, number, or other
sequence of characters which reads
the same backward or forward.

14
factor-independent (intrinsic)
221 S16