DNA

1
Gene expression the path from gene to protein
DNA
RNA
Transcription
Darst lab
RNA
Translation
Protein
Frank lab
Ribosome
2
Bacterial transcription cycle
?
?
Achillefs Kapanidis
3
ALTERNATING-LASER EXCITATION (ALEX) SPECTROSCOPY
Single-pair FRETSPECTROSCOPY
Ha et al, PNAS, 1996
Deniz et al, PNAS, 1999
Kapanidis et al, PNAS, 2004
4
ALEX assay for protein movement and dissociation
s70 release in elongation
LEADING-EDGE FRET ASSAY
Initiation Open complex
S
E
1
S
RNAP
S
D
A
s
0
1
0
E
S
5
Studying s70 retention in transcription
elongation Leading-edge FRET
2 nm distance change equivalent to RNAP
translocation by 1 turn of DNA
R7.9 nm
R5.9 nm
Open complex
E
70
RPo

• s70 retention in the first elongation complex
• High translocational activity

1
S
complex
S
0.5
DNA
0
1
0.5
0
150
E
Kapanidis et al., Mol. Cell, 20, 347 (2005)
6
Trailing-edge FRET
7
s70 Retention in early elongation - kinetics
s70 Retention in Early Elongation
8
Abortive initiation

• First described in 1980
• Important for
• regulation (can be rate-limiting step)
• promoter escape
• antimicrobial therapy using antibiotics (e.g.,
  rifampicin)
• Main observables during abortive initiation
• extension of downstream boundary of RNAP
  footprint
• upstream boundary appears fixed

9
Initial Transcription RNAP-active-center
translocation problem

• RPitc is able to synthesize RNA products up to
  8-10 nt in length (at some promoters, up to 15
  nt in length)

RNAP active center translocates in initial
transcription

• DNA-footprinting results indicate that the
  upstream boundary of the promoter DNA segment
  protected by RNAP is unchanged in RPitc vs. in
  RPo

RNAP appears not to translocate in initial
transcription
10
Initial Transcription RNAP-active-center
translocation proposed mechanisms
Kapanidis et al., Science 314, 1097 (2006 )
11
Detection of Initial Transcription
distance between RNAP leading edge and downstream
DNA
decreased distance
Kapanidis et al., Science 314, 1097 (2006 )
12
Test of Transient-Excursions Model
distance between RNAP trailing edge and upstream
DNA
Kapanidis et al., Science 314, 1097 (2006 )
13
Test of Inchworming Model
distance between RNAP leading edge and upstream
DNA
Kapanidis et al., Science 314, 1097 (2006 )
14
Test of Scrunching Model
distance between upstream DNA and downstream DNA
Kapanidis et al., Science 314, 1097 (2006 )
15
Map of distance changes during abortive initiation

• Data show DNA as flexible element in a pure
  DNA-scrunching mechanism

• Strain in DNA can drive promoter escape

RNAP
- Agreement with single-molecule DNA
nanomanipulation
Kapanidis et al., Science 314, 1097 (2006 )
16
Structural model
Kapanidis et al., Science 314, 1097 (2006 )
17
A working model for a promoter escape
bubble forms
bubble DNA scrunches stressed intermediate formed
scrunched DNA moves upstream
stress released by reforming dsDNA