Transcription in prokaryotes begins with RNA polymerase binding to DNA and transcribing genes. Bacterial genes have simple structures like promoters, Shine-Dalgarno sequences, and terminators. RNA polymerase initiates transcription at promoters and terminates at intrinsic terminator hairpin loops or extrinsic Rho-dependent sequences. Prokaryotic transcription can produce both monocistronic and polycistronic mRNAs.

1. Transcription in
Prokaryotes
M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

2.  Gene expression begins with
transcription
 RNA copy of a gene made by an RNA
polymerase
 Prokaryotic RNA polymerases are assemblies of
several different proteins

3. Bacterial genomes have simple gene structure
- Promoter
-35 sequence (T82T84G78A65C54A45) 15-20 bp
-10 sequence (T80A95T45A60A50T96) 5-9 bp (Pribnow Box)
- Start of transcription : initiation start: Purine90 (sometimes
it’s the “A” in CAT)
- Translation binding site (Shine-Dalgarno) 10 bp upstream of
AUG (AGGAGG)
- One or more Open Reading Frame
•start-codon (unless sequence is partial)
•until next in-frame stop codon on that strand ..
Separated by intercistronic sequences.
- Termination
Bacterial Gene: Structure of signals
Gene 1 Gene 2

4. RNA polymerase must know where the
start of a gene is in order to copy it
RNA polymerase has weak interactions
with the DNA unless it encounters a
promoter
 A promoter is a specific sequence of
nucleotides that indicate the start site for RNA
synthesis

5. General Steps of Transcription
 Initiation:
 Binding of RNA polymerase to double stranded DNA
 Development of closed promoter complex
 Development of open promoter complex
 Start of transcription by adding the first two
ribonucleotides.
 Elongation:
- Formation of transcription bubble or Transcription
elongation complex.
 Progression of the complex gradually in the
3’ direction to elongate the initiated RNA chain.
 Rapid process: up to 40 nucleotides per second.
 On the same gene there are several RNA strands being
transcribed in a staggered fashion.
 Termination:
 Terminator sequences signal stop of transcription.

6. Sigma
dissociates
Initiation

7. Irreversible form of Open Complex Formation

8. Generation of Abortive Initiation
Products

9. Transcription Bubble

10. RNA Elongation
 Reads template 3’ to 5’
 Adds nucleotides 5’ to 3’
(5’ phosphate to 3’
hydroxyl)
 Synthesis is the same as
the leading strand of DNA

11. Polymerization is polar: enzyme works by adding
to a free 3’ hydroxyl in growing mRNA chain.

12. RNA Synthesis
 RNA pol moves
nt by nt,
unwinds the
DNA as it goes
 Will stop when
it encounters a
STOP.
 RNA pol
leaves,
releasing the
RNA strand

13. Termination of Transcription
Factor-independent termination
Factor-dependent termination
– 3 factors
• Rho (ρ), Tau (τ) and NusA
– Rho best studied

14. Termination of transcription
RNA: single stranded nucleic acid
• can form secondary structures
Rho-dependent termination: protein signal
• Rho binds to RNA; able to cause RNA &
RNA polymerase to leave DNA
→ termination
Rho-independent signal: hairpin or stem-
loop RNA structure forms, followed by
several uracils
→ termination

15. Terminator Sequences
In prokaryotes there are two types:
1. Intrinsic: Rho (ρ) independent terminator
Contains a G-C rich region followed by six or more
A-T sequences.
 Causes the formation of a double stranded RNA
called a hairpin loop.
 Retards the movement of the RNA polymerase
along the DNA molecule, and causes
termination at the A-T rich region.

16. 2. Extrinsic: Rho-dependent terminator
Requires a protein factor called Rho (ρ).
Rho protein trails the RNA polymerase until
it reaches a GC rich region, when Rho
catches up with the polymerase.
Rho protein pulls off RNA from
transcription bubble.

17. 1. Rho-independent terminator site
RNA transcript at the terminating site is
self-complementary
The bases can pair to form a hairpin
structure with a stem and loop, a structure
favored by its high G-C content
The stable hairpin is followed by a
sequence of 4 or more U residues
The RNA transcript ends within or just
after them

18. Intrinsic termination site
String of Us
Intrastrand
complementary
bases

19. Mechanism of Rho-independent
Termination
 RNA polymerase pauses when it encounters
such a hairpin formed at the terminator site
 The RNA-DNA hybrid helix produced after
the hairpin is unstable because of its content
of rU-dA base pairs, the weakest of the four
kinds of pairs
 Nascent RNA is pulled off from the DNA
template and then from the enzyme
 DNA template strand now joins its partner to
form the DNA duplex

20. Control of trp operon by attenuation:
stalled translation allows region
2 to interact with region 3
3 & 4 cannot interact
regions 3 & 4 interact;
termination results

21. Global control systems inGlobal control systems in E. coliE. coli::
In global control systems: many genes, pathways regulated
simultaneously in response to a specific environmental signal
• e.g., regulon: collection of genes and/or operons controlled
by common regulatory protein
• Sporulation in Bacillus: another global control system:

22. 2. Rho-dependent terminator

23. Rho (ρ) Protein
 Rho is an RNA-dependent ATPase
 Also an RNA-DNA helicase
 It is an hexamer, with a mass of 275 kDa (each
subunit is of 46 kDa)
 It binds to ssRNA at Rut site – a stretch of 72 nt
is bound, 12 per subunit
 It is brought into action by sequence located in
the nascent RNA
 ATPase activity enables it to move
unidirectionally along the nascent RNA

24. Effect of rho protein on the size of
RNA transcripts

25. Rho factor: factor
mediated termination
 In an ATP-mediated
reaction, a rho protein
complex binds to the
mRNA and unwinds
RNA from the DNA
template
 Recognition sites
may not have hairpins
or U tracts; tend to be
C-rich
?
Rho-dependent:

27. One Transcriptional unit

28. Two contiguous genes
 RNA is released so we can make many
copies of the gene, usually before the first
one is done
 Can have multiple RNA polymerase molecules on
a gene at a time
Initiation
site
Termination
site
RNA fibrils

29. Summary

30. Types of mRNA
In Bacteria:
Monocistronic mRNA
Polycistronic mRNA
In Eukaryotes:
Monocistronic mRNA

31. Polycistronic mRNA
 Many prokaryotic mRNAs are polycistronic
Contain sequences specifying the synthesis of
several proteins
 A polycistronic mRNA molecule possesses a
series of start and stop codons
In case it codes for three proteins:
Start, Protein1, Stop – Start, Protein2, Stop
– Start, Protein3, Stop
 Abou 5-20 bases may be present between one
stop codon and the next start codon. These
are called Spacers.
 The segment of RNA corresponding to a DNA
cistron is called a Reading frame

32. PolycistronicPolycistronic vsvs Monocistronic mRNAMonocistronic mRNA
spacers

33. Processing of pre-rRNA
transcripts in E. coli