1. Transcription in Prokaryotes

2. Transcription: production of mRNA copy of the DNA gene.
Eukaryote model

3. Transcription RNA Not all RNA is translated into protein:
Some RNA is structural - e.g. ribosomal RNA (rRNA)
Some RNA is functional - e.g. transfer RNA (tRNA)
Some RNA is chromosomal (some viruses)
The production of protein-encoding RNA in bacteria is the subject of this lecture.

4. Transcription
From which DNA strand is RNA synthesized?
 Transcription usually takes place on only ONE of the DNA strands (though not necessarily the same strand throughout the entire chromosome).

5. 3'-CAGTGGGTACCTCC-5' Template strand 5'-GUCACCCAUGGAGG-3' mRNA
Transcription
RNA growth always in the 5'  3' direction
5'-GTCACCCATGGAGG-3' Nontemplate strand
3'-CAGTGGGTACCTCC-5' Template strand
5'-GUCACCCAUGGAGG-3' mRNA
mRNA 3' 5' 5' 3' 3' 5'
DNA 5' 3' 5' 3' 5' 3'
mRNA
mRNA
mRNA

6. Transcription
RNA Polymerase
The synthesis of RNA from a DNA template is carried out by enzymes known formally as DNA-dependent RNA polymerases, now simply referred to as RNA polymerases

7. 3'-CA CC-5‘ Template strand G T TGGGTACC
Transcription
ACCCATGG
C A
5'-GT GG-3' Nontemplate
“CODING”strand
CAUG C A
5'-GUC
3'-CA CC-5‘ Template strand
G T
TGGGTACC
RNA polymerase

8. RNA Polymerase RNA polymerases have the following properties:
 The enzymes are template dependent, requiring double-stranded DNA
 The enzymes require the four nucleoside triphosphates (ATP, GTP, CTP, and UTP)
 The enzymes copy (read) the template DNA strand in the 3' to 5' direction
 The enzymes synthesize the RNA in the 5' to 3' direction

9. Order of events in Transcription
Binding of polymerases to the initiation site, the promoter. Prokaryotic polymerases can recognise the promoter and bind to it directly.
Unwinding (melting) of the DNA double helix by a helicase. In prokaryotes the polymerase has the helicase activity.
Synthesis of RNA based on the sequence of the DNA template strand, using nucleoside triphosphates (NTPs) to construct RNA.
4) Termination of synthesis. NOTE: the “STOP” codon in the genetic code for the end of peptide synthesis is NOT the end of termination.

10. Prokaryotic RNA Polymerase: Core Enzyme  
Chain initiation and interaction with regulatory proteins
Catalytic center: chain initiation and elongation
DNA binding

11. RNA Polymerase
 The core enzyme has the ability to synthesize RNA, however, the initiation point of RNA synthesis is non-specific.
 An additional subunit, the sigma factor, is required to initiate RNA synthesis at specific locations in the DNA, termed the promoter.

12. Prokaryotic RNA Polymerase: Holoenzyme
 Promoter recognition

13. Promoters
For any given gene, RNA synthesis always starts at the same point on the DNA, the promoter. What is a promoter?
Hypothesis: Because one RNA polymerase copies every gene and binds to the promoter in each gene to do so, the promoters in different genes must have similarities. Similarities in DNA must lie in the sequence of nucleotides so the promoters of every gene must have the same sequence of nucleotides.

14. Promoters
David Pribnow tested this by comparing the sequences in the promoter regions of five genes from E. coli. He found a conserved sequence of nucleotides in each. This was called the Pribnow box.
Pribnow

15. Promoters
The Pribnow box lies 10 nucleotides from the transcription start point (TSP). A second was later found 80 nucleotides away.
TSP
TTGACA TATAAT
DNA
Pribnow box 5' 3' 3' 5'
RNA

16. Promoters
Consensus sequences
The sequences found in promoters are to some extent imaginary. Very few genes actually contain these sequences but they all contain a sequence that is only a few nucleotides different. The consensus sequence is a “best average”.

17. Promoters GCGTTGTCATGC gene1 AATGTGACAGCT gene2 TGCTAGACACAG gene3
GAATTGAGAAAA gene4
CTTTTCACATTC gene5
AGCTAGACAGGG gene6
TCGTTGGCACCA gene7
CCAATGACCATT gene8
ATGTTGACTTGC gene9
TTGACA consensus not actually in any of the genes

18. Promoters
Just because consensus sequences have been found, this doesn’t mean that they are functional. What is the evidence that they actually work?

19. Promoters
Although sequences can vary from the consensus, some mutations stop the promoter from working. In these cases, it demonstrates that the consensus sequence is a functional promoter.
Genes that are transcribed strongly have sequences more like the ideal consensus than genes that are transcribed weakly.

20. Transcription
RNA polymerase scans DNA double helix, searching for a promoter site.
Promoter region in DNA
RNA polymerase

21. Initiation (1) Sigma binds to promoter region.
Sigma residues Y425, Y430 and W434 directly involved in the unwinding (melting) of the double helix.

22. Initiation
Sigma binds to promoter region, recognizing both the -35 and -10 regions. The resulting structure is termed a closed promoter complex.
The promoter is rich in A and T. 
The AT pair involves two hydrogen bonds whereas the CG pair involves three hydrogen bonds.  Therefore, AT pairs are easier to separate.

23. Initiation
(2) After the DNA strands have been separated at the promoter region by the helicase activity of the sigma subunit, forming an open promoter complex. The core subunit () can then start to synthesize RNA.
(3) Following initiation, the sigma subunit is released after approx. 10 ribonucleotides have been polymerized,

24. Elongation
Synthesis of the RNA strand continues until the core polymerase reaches the termination site.

25. Termination
In prokaryotes, the transcription is terminated by two major mechanisms:
Rho-independent (intrinsic) and
Rho-dependent.

26. Termination
Rho-independent
The Rho-independent termination signal is a stretch of bp sequence, consisting of many GC residues followed by a series of T ("U" in the transcribed RNA). 
The resulting RNA transcript will form a stem-loop structure to terminate transcription

27. The terminator has the following structure:
Termination
The terminator has the following structure:
Complementary
GC rich GC rich PolyA
DNA
RNA
GC rich GC rich PolyU

28. Termination stem-loop structure U C U G G C A U C G GC rich regions
Poly U
RNA
UAAUCCCACAG CAUUUU

29. Termination
TAG GC rich 1 GC rich 2 ATrich 5 3 5 3
As transcription proceeds, the two GC rich regions base pair. This leaves a short poly U rich region, which cannot pair strongly enough to hold the RNA onto the DNA. The polymerase comes off with it.
RNA polymerase
RNA

30. Termination Rho-dependent.
In vitro, E. coli RNA polymerase holoenzyme transcribes DNA into a very long RNA.
The ability of the in vitro reaction to make natural length RNA is restored by the addition of a protein factor, called rho ().
RNA transcript length:
By holo polymerase in vitro
In vivo
By holo polymerase + rho

31. Termination
Analysis of termination sites dependent on rho revel a stem loop structure near the 3‘ end of the RNA, with NO U-rich tale.
Rho binds to RNA and can, if provided with ATP, move along the RNA.
Rho also has ATP-dependent helicase activity.

32. Model for rho termination
It has been established that six Rho proteins form a hexamer to terminate transcription, but the precise mechanism is not clear.
(1) The Rho hexamer first binds to the RNA transcript at an upstream site which is nucleotides long and rich in C residues .

33. Termination
(2) Upon binding, the Rho hexamer moves along the RNA in the 5-3 direction, trying to catch up with the RNA polymerase.
(3) When the polymerase pauses, which happens when secondary structures form near the 3 end of the RNA, rho catches up and melts the RNA-DNA duplex in the replication bubble, causing termination.

34. Transcription and translation
Termination
DNA
Ribosomes
Transcription and translation
RNA
Protein

35. Ribosome dissociates from the RNA when they encounter a stop codon.
Termination
DNA
UAG
Ribosome dissociates from the RNA when they encounter a stop codon.

36. Rho factor binds to specific sites on naked RNA.
Termination
DNA
Rho factor binds to specific sites on naked RNA.
UAG
(i.e. RNA without ribosomes)

37. RNA polymerase pauses at stem loop, while rho moves along RNA, 5-3.
Termination
DNA
RNA polymerase pauses at stem loop, while rho moves along RNA, 5-3.
UAG

38. Termination
Rho catches up with polymerase, melting RNA-DNA duplex, causing polymerase to dissociate.
DNA
UAG

39. Termination
Termination of transcription can serve a role in regulating gene expression in prokaryotes
This is the subject of the final lecture in this series.

40. Suggested reading http://www.nottingham.ac.uk/bennett-lab/lee.html
Transcription (2000) In: An Introduction to Genetic Analysis. pp Griffiths, A. J. F,. Miller, J. H., Suzuki, D. T., Lewontin, R. C. and Gelbart, W. M. (Eds). Freeman and Company, New York.