1. Transcription in Prokaryotes

2. Transcriptional Control
Environmental change
Turn gene(s) on/off
Proteins to deal with
new environment
DNA
RNA
protein
Very important to:
express genes when needed
repress genes when not needed
Conserve energy resources; avoid expressing unnecessary/detrimental genes

3. Transcriptional Control
Many places for control
Transcription
Initiation
Elongation
Termination
Processing
Capping
Splicing
Polyadenylation
Turnover
Translation
Protein processing
DNA
RNA
protein

4. Prokaryotic Transcription
Operons
Groups of related genes transcribed
by the same promoter
Polycistronic RNA
Multiple genes transcribed
as ONE TRANSCRIPT
No nucleus, so transcription and
translation can occur simultaneously

5. RNA Structure Contain ribose instead of deoxyribose Bases are A,G,C,U,
Uracil pairs with adenine
Small chemical difference from DNA, but large structural differences
Single stranded helix
Ability to fold into 3D shapes - can be functional

6. RNA Structures Vary RNA more like proteins than DNA:
structured domains connected by more flexible domains, leading to different functions
e.g. ribozymes – catalytic RNA

7. RNA synthesis
RNAP binds, melts DNA
Nucleosides added 5’  3’

8. Types of RNA Messenger RNA (mRNA) – genes that encode proteins
Ribosomal RNA (rRNA) – form the core of ribosomes
Transfer RNA (tRNA) – adaptors that link amino acids to mRNA during translation
Small regulatory RNA – also called non-coding RNA

9. Transcriptional Control
Initiation
Elongation
Termination
Processing
Capping
Splicing
Polyadenylation
Turnover
Translation
Protein processing
Control of initiation
usually most
important.

10. Initiation RNA polymerase Transcription factors Promoter DNA
RNAP binding sites
Operator – repressor binding
Other TF binding sites
Start site of txn is +1
α α β β’σ

11. Initiation RNA polymerase 4 core subunits Sigma factor (σ)–
determines promoter
specificity
Core + σ = holoenzyme
Binds promoter sequence
Catalyzes “open complex” and transcription of DNA to RNA

12. RNAP binds specific promoter sequences
Sigma factors recognize consensus
-10 and -35 sequences

13. RNA polymerase promoters
TTGACA
TATAAT
Deviation from consensus -10 , -35 sequence leads to
weaker gene expression

14. Bacterial sigma factors
Sigma factors are “transcription factors”
Different sigma factors bind RNAP and recognize specific -10 ,-35 sequences
Helps melt DNA to expose transcriptional start site
Most bacteria have major and alternate sigma factors
Promote broad changes in gene expression
E. coli 7 sigma factors
B. subtilis 18 sigma factors
Generally, bacteria that live in more varied environments have more sigma factors

15. Sigma factors E. coli can choose between 7 sigma factors and about 350sS sS sF
s32
Extreme heat shock, unfolded proteins
E. coli can choose between 7 sigma factors and about 350
transcription factors to fine tune its transcriptional output
An Rev Micro Vol. 57: T. M. Gruber

16. What regulates sigma factors
Number of copies per cell (σ70 more than alternate)
Anti-sigma factors (bind/sequester sigma factors)
Levels of effector molecules
Transcription factors

17. Bacterial RNAP numbers
In log-phase E. coli:
~4000 genes
~2000 core RNA polymerase molecules
~2/3 (1300) are active at a time
~1/3 (650) can bind σ subunits.
Competition of σ for core determines much of a cell’s protein content.

18. Lac operon control Repressor binding prevents RNAP binding promoter
An activating transcription factor found to be
required for full lac operon expression: CAP (or Crp)

19. lac operon – activator and repressor
CAP = catabolite
activator protein
CRP = cAMP receptor
protein

20. Activating transcription factors
Crp dimer w/ DNA
Helix-turn-helix (HTH) bind major groove
of DNA
HTH one of many
TF motifs

21. Cofactor binding alters conformation
Crp binds cAMP, induces allosteric changes
glucose
cAMP
Crp
lac operon
no mRNA
glucose
cAMP
Crp
mRNA

22. Cooperative binding of Crp and RNAP
Binds more stably than either protein alone

23. Enhancers binding site NtrC example: NtrC required for RNAP to
activating regions not
necessarily close to RNAP
binding site
NtrC example:
NtrC required for RNAP to
form open complex
NtrC activated by P
P NtrC binds DNA, forms loop
that folds back onto RNAP,
initiating transcription
signature of sigma 54

24. Bacterial promoters Most bacterial promoters have –35 and –10 elements
Transcription start +1
UP element
-35 element
-10 element (Pribnow box) +1
pre –10 element
Most bacterial promoters have –35 and –10 elements
Some have UP element
Some lack –35 element, but have extended –10 region

25. E. coli RNA polymerase composed of 5 subunits:
Subunits: b, b’, a(2), , and s
Core enzyme: b, b’, a(2), 
Holoenzyme : b, b’, , a(2) and s
The s subunits give specificity for site of initiation- promoter

26.  ’  s NTD CTD Subunit structure of bacterial RNA polymerase 160 kDa
DNA
Holoenzyme-b’ba2s. Functions in initiation.
Core enzyme-b’ba2. Functions in elongation.

27. The 3D structure of bacterial RNA polymerase holoenzyme
s factor domains :
N-term s1
Inhibition s2
-10 binding s3
-10 binding s4
-35 binding

28. The s factors
s factors are required for promoter recognition and transcription initiation in prokaryotes
s factors have analogous function as general transcription factors in eukaryotes
A variety of s factors exist in E.coli
For expression from most promoters s70 is required
For expression from some bacterial promoters one of other s subunits is needed instead
s70 is essential for cell growth in all conditions, while other sigmas are required for special events, like nitrogen regulation (s54), response to heat shock (s32), sporulation, etc

29. The overview of s factor function
RNA pol
Holoenzyme
Promoter region
Closed complex
Open complex
Promoter escape
Elongation
mRNA
s release
The overview of s factor function

30. The promoter specificity of some s factors in E.coli
s70 TTGACA – 17 bp – TATAATN3-6-A
s CTTGAAA – 16 bp – CCCCATNTN3-10-T/A
s GG – N12 – GC/T – 12bp – A

31. The UP element
RNAP
RNAP
a NTD s
a CTD s4
s2-3 UP
-35
-10 +1
UP element is an AT rich motif present in some strong (e.g. rRNA) promoters
UP element interacts directly with C-terminal domain of RNA polymerase a subunits

32. Constitutive and inducible promoters
Certain genes are transcribed at all times and circumstances
-Examples – tRNAs, rRNAs, ribosomal proteins, RNA polymerase
-Promoters of those genes are called constitutive
Most genes, however, need to be transcribed only under certain circumstances or periods in cell life cycles
-The promoters of those genes are called inducible and they are subject to up- and down- regulation

33. Regulation at promoters
Promoters can be regulated by repression and/or activation
Many s70 promoters are controlled both by repression and activation, whereas, for example s54 promoters are controled solely by activation

34. Cartoon of the transcription cycle

35. Mechanisms of repression
Repression by steric hindrance
Inhibition of transition to open complex
Inhibition of promoter clearance
Anti-activation
Anti-sigma factors

36. e) Anti-sigma factors
An anti-s factor is defined by the ability to prevent its cognate s factor to compete for core RNA polymerase
Mostly used for s factors, other than s70, for example in life cycle regulation (sporulation, etc)
Some bacteriophages use their own anti-s factors to prevent expression of cellular proteins
RNAP
RNAP
anti-s s s

37. Repressor molecule removes the activator
d) Anti-activation
Repressor molecule removes the activator
RNA pol - s
Activator
ABS
weak promoter +1
Activator binding sequence
Activator
RNA pol - s
Repressor
ABS
weak promoter +1

38. Two examples of steric hindrance
Trp repressor
Lac repressor

39. The tryptophan repressor
The trp repressor controls the operon for the synthesis of L-tryptophan in E.coli by a simple negative feedback loop
When enough tryptophane (blue dots) is made, it binds to repressor, which now is able to bind to promoter and block RNA polymerase binding
In the absence of tryptophane the trp repressor (red blob) shows no affinity to promoter (black box) and the RNA polymerase (yellow blob) transcribes the operon

40. The lac promoter
Lac promoter is widely used in artifical plasmids, designed for protein production
For practical purposes it is easier to use non-hydrolyzable lactose analog – IPTG (isopropyl-b-thiogalactoside) instead of native lactose

41. A cartoon, ilustrating events upon IPTG binding to lac repressor
As IPTG binds, the DNA binding domains scissor apart

42. Mechanisms of activation
a) Regulated recruitment
b) Polymerase activation
c) Promoter activation

43. a) Regulated recruitment
Activator “extends” the binding site for RNA polymerase
strong or weak affinity
RNA pol - s
Activator
ABS
weak promoter +1
strong affinity
weak affinity

44. Catabolite Activator Protein: CAP
Activates transcription from more than 150 promoters in E.coli
Upon activation by cAMP (cyclic Adenosine MonoPhosphate), CAP binds to promoter and helps RNAP-s to bind as well
All CAP–dependent promoters have weak –35 sequence, so that RNAP-s is unable to bind the promoter without CAP assistance

46. Models for Class I and Class II promoter activation
Class I CAP binding sites can be from –62 to –103. CAP interacts with the carboxy terminal domain of the RNAP a-subunit (aCTD)
Class II CAP binding sites usually overlap the –35. CAP interacts with the aCTD, aNTD (N-terminal domain), and the s factor
Busby and Ebright, 2000, J. Mol. Biol. 293:

47. Model for Class III promoter activation
Activation of Class III promoters requires binding of at least two CAP dimers or at least one CAP dimer and one regulation-specific activator
Interactions can be similar to those of ClassI and/or ClassII promoters, except that each aCTD subunit is making different interactions

48. AraC – repressor and activator of arabinose promoter
RNAP-s
Transcription
+ arabinose ( )
AraC
promoter
DNA binding domain of AraC

49. RNAP-s54 activation
ATP+Pi
ATP
s54
s54
RNAP-s54 open complex formation requires ATP hydrolysis
Activator protein with ATP-ase activity binds to “enhancer” site about 160 bp upstream from –24 sequence. DNA then gets looped and activator interacts with RNAP-s54 resulting in the open bubble formation upon ATP hydrolysis

50. c) Example of promoter activation: MerR activator family
MerR is an activator that controls genes involved in the response to mercury poisoning
Other MerR family activators (CueR, BmrR, etc) respond to a variety of different toxic compounds such as other heavy metal atoms or drugs
In MerR activated promoters, -10 and –35 regions are separated by 19bp instead of optimal 17bp

51. DNA-protein interaction assays
Electrophoretic mobility shift assay (EMSA)
DNase I Footprinting
Chromatin immunoprecipitation (ChIP)

52. EMSA Radiolabel promoter sequence
Incubate one sample with cell lysates or purified protein and the other without
TF will bind promoter sequence
TF-bound probe
Run DNA-protein mixture on polyacrylamide gel and visualize w/ audoradiography
Free probe

53. EMSA CovR PcylE CovR DNA binding protein Binds to cylE promoter
Recognition sequence ‘TATTTTAAT’
CovR
PcylE

54. DNase I Footprinting
Method to determine where a protein binds a DNA sequence

55. DNase I footprint 1 -- DNA sequence ladder 2 -- DNA sequence ladder
3 -- No protein
4 -- (+) RNA polymerase
5 -- (+) lac repressor

56. ChIP Crosslink proteins bound to DNA
Immunoprecipitate lysate for specific transcription factor, RNAP, etc
Analyze DNA bound to protein by PCR

57. Transcriptional Control
Initiation
Elongation
Termination
Processing
Capping
Splicing
Polyadenylation
Turnover
Translation
Protein processing

58. Transcriptional Termination
Bacteria need to end transcription at the end of the gene
2 principle mechanisms of termination in bacteria:
Rho-independent (more common)
Rho-dependent

59. Rho-independent termination
Termination sequence has 2 features:
Series of U residues
GC-rich self-complimenting region
GC-rich sequences bind forming stem-loop
Stem-loop causes RNAP to pause
U residues unstable, permit release of RNA chain

60. Rho-dependent termination
Rho is hexameric protein
70-80 base segment of RNA wraps around
Rho has ATPase activity, moves along RNA until site of RNAP, unwinds DNA/RNA hybrid
Termination seems to depend on Rho’s ability to “catch up” to RNAP
No obvious sequence similarities, relatively rare

61. Transcriptional attenuation
Attenuator site = DNA sequence where RNAP chooses between continuing transcription and termination
trp operon
4 RNA regions
for basepairing
2 pairs w/ 1 or 3
3 pairs w/ 2 or 4
Concentration of
Trp-tRNATrp determines
fate of attenuation
At high Trp conc,
transcription stops via
Rho-independent

62. Anti-termination
λ phage encode protein that prevents termination

63. Two Component Systems

64. Two Component Systems
‘Histidine kinase’ senses environmental changes- autophosphorylates at conserved histidine residue
Response regulator is phosphorylated by activated sensor kinase at conserved aspartate- activates or represses transcription/function
Way for bacteria to sense environmental changes and alter gene expression

65. Quorum Sensing
Bacteria produce and secrete chemical signal molecules (autoinducers)
Concentration of molecules increases with increasing bacterial density
When critical threshold concentration of molecule is reached, bacteria alter gene expression
Way for communities of bacteria to “talk” to each other

66. Quorum Sensing in Vibrio fischeri
at high cell density, V. fischeri
express genes for bioluminescence
LuxI produces autoinducer
acyl-homoserine lactone
AHL diffuses outside of cell
when AHL reaches critical
concentration, it binds LuxR
activated LuxR bound AHL
activates transcription of
luminescence genes

67. Transcription termination
In prokaryotes two types of transcription termination occur – rho indepedent termination and rho dependent termination
In rho independent case, the termination is achieved by a secondary structure of mRNA – RNA stem-loop, followed by an AU rich region
A rho protein is required for rho-dependent termination

68. Rho independent termination

69. Attenuation Regulation of transcription by the behavior of ribosomes
Observed in bacteria, where transcription and translation are tightly coupled
Translation of a mRNA can occur as the mRNA is being synthesized

70. Attenuation in trp operon

71. Rho dependent termination
As polymerase transcribes away from the promoter, rho factor binds to RNA and follows the polymerase
When polymerase reaches some sort of pause site, rho factor catches up with polymerase and unwinds the DNA-RNA hybrid, resulting in release of polymerase

72. Anti-termination