1. Molecular Biology Fifth Edition
Lecture PowerPoint to accompany
Molecular Biology Fifth Edition
Robert F. Weaver
Chapter 6
The Mechanism of Transcription in Bacteria
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

2. 6.1 RNA Polymerase Structure
By 1969 SDS-PAGE of RNA polymerase from E. coli had shown several subunits
2 very large subunits are b (150 kD) and b’ (160 kD)
Sigma (s) at 70 kD
Alpha (a) at 40 kD – 2 copies present in holoenzyme
Omega (w) at 10 kD
Was not clearly visible in SDS-PAGE, but seen in other experiments
Not required for cell viability or in vivo enzyme activity
Appears to play a role in enzyme assembly

3. Sigma as a Specificity Factor
Core enzyme without the s subunit could not transcribe viral DNA, yet had no problems with highly nicked calf thymus DNA
With  subunit, the holoenzyme worked equally well on both types of DNA

4. Summary The key player in the transcription process is RNA polymerase
The E. coli enzyme is composed of a core, which contains the basic transcription machinery, and a -factor, which directs the core to transcribe specific genes

5. 6.2 Promoters
Why was the core RNA polymerase capable of transcribing nicked DNA in the previous table?
Nicks and gaps are good sites for RNA polymerase to bind nonspecifically
The presence of the s-subunit permits recognition of authentic RNA polymerase binding sites called promoters
Transcription that begins at promoters is specific, directed by the s-subunit

6. Binding of RNA Polymerase to Promoters
How tightly does core enzyme v. holoenzyme bind DNA?
Experiment measures binding of DNA to enzyme using nitrocellulose filters
Holoenzyme binds filters tightly
Core enzyme binding is more transient

7. Temperature and RNA Polymerase Binding
As the temperature is lowered, the binding of RNA polymerase to DNA decreases dramatically
Higher temperatures promote DNA melting and encourage RNA polymerase binding

8. RNA Polymerase Binding
Hinkle and Chamberlin proposed:
RNA polymerase holoenzyme binds DNA loosely at first
Binds at promoter initially
Scans along the DNA until it finds a promoter
Complex with holoenzyme loosely bound at the promoter is a closed promoter complex as DNA is in a closed ds form
Holoenzyme can then melt a short DNA region at the promoter to form an open promoter complex with polymerase bound tightly to DNA

9. Polymerase/Promoter Binding
Holoenzyme binds DNA loosely at first
Complex loosely bound at promoter = closed promoter complex, dsDNA in closed form
Holoenzyme melts DNA at promoter forming open promoter complex - polymerase tightly bound

10. Summary
The -factor allows initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoter
This tight binding depends on local melting of the DNA to form an open promoter complex and is stimulated by 
The -factor can therefore select which genes will be transcribed

11. Core Promoter Elements
There is a region common to bacterial promoters described as 6-7 bp centered about 10 bp upstream of the start of transcription = -10 box
Another short sequence centered 35 bp upstream is known as the -35 box
Comparison of thousands of promoters has produced a consensus sequence (or most common sequence) for each of these boxes

12. Promoter Strength Consensus sequences:
-10 box sequence approximates TATAAT
-35 box sequence approximates TTGACA
Mutations that weaken promoter binding:
Down mutations
Increase deviation from the consensus sequence
Mutations that strengthen promoter binding:
Up mutations
Decrease deviation from the consensus sequence

13. UP Element
The UP element is upstream of the core promoter, stimulating transcription by a factor of 30
UP is associated with 3 “Fis” sites which are binding sites for the transcription-activator protein Fis, not for the polymerase itself

14. The rrnB P1 Promoter Transcription from the rrn promoters respond
Positively to increased concentration of iNTP
Negatively to the alarmone ppGpp

15. 6.3 Transcription Initiation
Transcription initiation was assumed to end as RNA polymerase formed 1st phosphodiester bond
Carpousis and Gralla found that very small oligonucleotides (2-6 nt long) are made without RNA polymerase leaving the DNA
Abortive transcripts such as these have been found up to 10 nt

16. Stages of Transcription Initiation
Formation of a closed promoter complex
Conversion of the closed promoter complex to an open promoter complex
Polymerizing the early nucleotides – polymerase at the promoter
Promoter clearance – transcript becomes long enough to form a stable hybrid with template

17. Sigma Stimulates Initiation of Transcription
In this first experiment stimulation by s appears to cause both initiation and elongation
Or stimulating initiation by s provides more initiated chains for core polymerase to elongate
Further experiments by the same group proved that s does not stimulate elongation

18. Reuse of s
During initiation s can be recycled for additional use with a new core polymerase
The core enzyme can release s which is then free to associate with another core enzyme

19. Fluorescence Resonance Energy Transfer
The -factor changes its relationship to the core polymerase during elongation
It may not dissociate from the core but actually shift position and become more loosely bound to core
To answer this question Fluorescence Resonance Energy Transfer (FRET) was used as it relies on two fluorescent molecules that are close enough together to engage in transfer of resonance energy
FRET allows the position of s relative to a site on the DNA to be measured without using separation techniques that might displace s from the core enzyme

20. FRET Assay for s Movement Relative to DNA

21. Models for the -Cycle
The obligate release version of the -cycle model arose from experiments performed by Travers and Burgess that proposed the dissociation of  from core as polymerase undergoes promoter clearance and switches from initiation to elongation mode
The stochastic release model proposes that  is indeed released from the core polymerase but that there is no discrete point of release during transcription and that the release occurs at random - a preponderance of evidence favors this model

22. Local DNA Melting at the Promoter
From the number of RNA polymerase holoenzymes bound to DNA, it was calculated that each polymerase caused a separation of about 10 bp
In another experiment, the length of the melted region was found to be 12 bp
Later, size of the DNA transcription bubble in complexes where transcription was active was found to be bp

23. Experiment to locate the region of early promoter melted by RNA Polymerase

24. Promoter Clearance
RNA polymerases have evolved to recognize and bind strongly to promoters
This poses a challenge when it comes time for promoter clearance as those strong bonds must be broken in order for polymerase to leave the promoter and enter the elongation phase

25. Promoter Clearance Several hypotheses have been proposed
The polymerase cannot move enough downstream to make a 10-nt transcript without doing one of three things:
- transient excursion: moving briefly downstream and then snapping back to the starting position
- inchworming: stretching itself by leaving its trailing edge in place while moving ots leading edge downstream
- scrunching: compressing the DNA without moving itself

26. Abortive Transcription, Scrunching and Promoter Clearance
Ebert and colleagues performed several experiments to distinguish between the hypotheses
Using E.coli polymerase the authors concluded that approximately 100% of all transcription cycles involved scrunching, which suggested that scrunching is required for promoter clearance
The E.coli polymerase achieves abortive transcription by scrunching: drawing downstream DNA into the polymerase without actually moving and losing its grip on promoter DNA
The scrunched DNA could store enough energy to allow the polymerase to break its bonds to the promoter and begin productive transcription

27. Structure and Function of s
Genes encoding a variety of s-factors have been cloned and sequenced
There are striking similarities in amino acid sequence clustered in 4 regions
Conservation of sequence in these regions suggests important function
All of the 4 sequences are involved in binding to core and DNA

28. Homologous Regions in Bacterial s Factors

29. E. coli s70 Four regions of high sequence similarity are indicated
Specific areas that recognize the core promoter elements are the -10 box and –35 box

30. Region 1
Role of region 1 appears to be in preventing s from binding to DNA by itself
This is important as s binding to promoters could inhibit holoenzyme binding and thereby inhibit transcription
Region 2
This region is the most highly conserved of the four
There are four subregions – 2.1 to 2.4
2.4 recognizes the promoter’s -10 box
The 2.4 region appears to be a-helix

31. Regions 3 and 4 Region 3 is involved in both core and DNA binding
Region 4 is divided into 2 subregions
This region seems to have a key role in promoter recognition
Subregion 4.2 contains a helix-turn-helix DNA-binding domain and appears to govern binding to the -35 box of the promoter

32. Summary
Comparison of different s gene sequences reveals 4 regions of similarity among a wide variety of sources
Subregions 2.4 and 4.2 are involved in promoter -10 box and -35 box recognition
The s-factor by itself cannot bind to DNA, but DNA interaction with core unmasks a DNA-binding region of s
Region between amino acids 262 and 309 of b’ stimulates s binding to the nontemplate strand in the -10 region of the promoter

33. Role of a-Subunit in UP Element Recognition
RNA polymerase itself can recognize an upstream promoter element, UP element
While s-factor recognizes the core promoter elements, what recognizes the UP element?
It appears to be the a-subunit of the core polymerase

34. Modeling the Function of the C-Terminal Domain
RNA polymerase binds to a core promoter via its s-factor, no help from C-terminal domain of a-subunit
Binds to a promoter with an UP element using s plus the a-subunit C-terminal domains (CTD)
Results in very strong interaction between polymerase and promoter
This produces a high level of transcription

35. 6.4 Elongation
After transcription initiation is accomplished, core polymerase continues to elongate the RNA
Nucleotides are added sequentially, one after another in the process of elongation

36. Function of the Core Polymerase
Core polymerase contains the RNA synthesizing machinery
Phosphodiester bond formation involves the b- and b’-subunits
These subunits also participate in DNA binding
Assembly of the core polymerase is a major role of the a-subunit

37. Role of b in Phosphodiester Bond Formation
Core subunit b lies near the active site of the RNA polymerase
This active site is where the phosphodiester bonds are formed linking the nucleotides
The s-factor may also be near the nucleotide-binding site during the initiation phase

38. Structure of the Elongation Complex
This section will examine how well predictions have been borne out by structural studies
How does the polymerase deal with problems of unwinding and rewinding templates?
How does it move along the helical template without twisting RNA product around the template?

39. RNA-DNA Hybrid
The area of RNA-DNA hybridization within the E. coli elongation complex extends from position –1 to –8 or –9 relative to the 3’ end of the nascent RNA
In T7 the similar hybrid appears to be 8 bp long

40. Structure of the Core Polymerase
X-ray crystallography on the Thermus aquaticus RNA polymerase core reveals an enzyme shaped like a crab claw
It appears designed to grasp the DNA
A channel through the enzyme includes the catalytic center
Mg2+ ion coordinated by 3 Asp residues
Rifampicin-binding site

41. Structure of the Holoenzyme-DNA Complex
Crystal structure of T. aquaticus holoenzyme-DNA complex as an open promoter complex reveals:
DNA is bound mainly to -subunit
Interactions between amino acids in region 2.4 of  and -10 box of promoter are possible
3 highly conserved aromatic amino acids are able to participate in promoter melting as predicted
2 invariant basic amino acids in  predicted to function in DNA binding are positioned to do so
A form of the polymerase that has 2 Mg2+ ions

42. Structure of the Elongation Complex
The X-ray crystal structure of the Thermus thermophilus RNA polymerase elongation complex in 2007 revealed several important observations
a valine residue in the E’ subunit inserts into the minor groove of the downstream DNA
the downstream DNA is double-stranded up to and including the +2 bse pair
the enzyme can accommodate nine base pairs of RNA-DNA hybrid
the RNA product in the exit channel is twisted into the shape it would assume as 1/2 of an A-form dsRNA

43. Topology of Elongation
Elongation of transcription involves polymerization of nucleotides as the RNA polymerase travels along the template DNA
Polymerase maintains a short melted region of template DNA
DNA must unwind ahead of the advancing polymerase and rewind behind it
Strain introduced into the template DNA ahead of the transcription bubble is relaxed by topoisomerases

44. Pausing and Proofreading
RNA polymerase frequently pauses, or even backtracks, during elongation
Pausing allows ribosomes to keep pace with the RNA polymerase, and it is the first step in termination
Backtracking aids proofreading by extruding the 3’-end of the RNA out of the polymerase, where misincorporated nucleotides can be removed by an inherent nuclease activity of the polymerase, stimulated by auxiliary factors

45. 6.5 Termination of Transcription
When the polymerase reaches a terminator at the end of a gene it falls off the template and releases the RNA
There are 2 main types of terminators
Intrinsic terminators function with the RNA polymerase by itself without help from other proteins
Other type depends on auxiliary factor called rho (r, these are rho or r-dependent terminators

46. Rho-Independent Termination
Intrinsic or rho-independent termination depends on terminators of 2 elements:
Inverted repeats followed immediately by
T-rich region in the nontemplate strand of the gene
An inverted repeat predisposes a transcript to form a hairpin structure due to complementary base pairing between the inverted repeat sequences

47. Inverted Repeats and Hairpins
The repeat at right is symmetrical around its center shown with a dot
A transcript of this sequence is self-complementary
Bases can pair up to form a hairpin as seen in the lower panel

48. Structure of an Intrinsic Terminator
Attenuator contains a DNA sequence that causes premature termination of transcription
The E. coli trp attenuator was used to show:
Inverted repeat allows a hairpin to form at transcript end
String of T’s in nontemplate strand result in weak rU-dA base pairs holding the transcript to the template strand

49. Model of Intrinsic Termination
Bacterial terminators act by:
Base-pairing of something to the transcript to destabilize RNA-DNA hybrid
Causes hairpin to form
This causes transcription to pause
a string of U’s incorporated just downstream of hairpin to destabilize the hybrid and the RNA falls off the DNA template

50. Rho-Dependent Termination
Rho caused depression of the ability of RNA polymerase to transcribe phage DNAs in vitro
This depression was due to termination of transcription
After termination, polymerase must reinitiate to begin transcribing again

51. Rho Affects Chain Elongation
There is little effect of rho or r on transcription initiation, if anything it is increased
The effect of rho or r on total RNA synthesis is a significant decrease
This is consistent with action of rho or r to terminate transcription forcing time-consuming reinitiation

52. Rho Causes Production of Shorter Transcripts
Synthesis of much smaller RNAs occurs in the presence of rho or r compared to those made in the absence
To ensure that this due to r itself and not to RNase activity of r, RNA was transcribed without r and then incubated in the presence of r
There was no loss of transcript size, so no RNase activity in r

53. Rho Releases Transcripts from the DNA Template
Compare the sedimentation of transcripts made in presence and absence of r
Without r, transcripts cosedimented with the DNA template – they hadn’t been released
With r present in the incubation, transcripts sedimented more slowly – they were not associated with the DNA template
It appears that r serves to release the RNA transcripts from the DNA template

54. Mechanism of Rho
No string of T’s in the r-dependent terminator, just inverted repeat to hairpin
Binding to the growing transcript, r follows the RNA polymerase
It catches the polymerase as it pauses at the hairpin
Releases transcript from the DNA-polymerase complex by unwinding the RNA-DNA hybrid

55. Summary
Using the trp attenuator as a model rho-independet terminator revealed two important features:
1 - an inverted repeat that allows a hairpin to for at the end of the transcript
2 - a string of T’s in the nontemplate strand that results in a string of weak rU-dA base pairs holding the transcript to the template strand
Rho-dependent terminators consist of an inverted repeat, which can cause a hairpin to form in the transcript but no string of T’s