DNA transcription is a process that involves transcribing genetic information from DNA to RNA. The transcribed DNA message, or RNA transcript, is used to produce proteins. DNA is housed within the nucleus of our cells. It controls cellular activity by coding for the production of proteins. The information in DNA is not directly converted into proteins, but must first be copied into RNA. This ensures that the information contained within the DNA does not become tainted.

1. Flow of Genetic Information
Recombination
Mutation/Repair

2. Three Different Classes of RNA
1) rRNA (ribosomal)
• large (long) RNA molecules
• structural and functional components of
ribosomes
• highly abundant
2) mRNA (messenger)
• typically small (short)
• encode proteins
• multiple types, not abundant
3) tRNA (transfer) and small ribosomal RNAs
• very small
• Important in translation
Not all genes encode proteins

3. In Bacteria
- all three classes are transcribed by the
same RNA polymerase
In Eukaryotes
- each class is transcribed by a different
RNA Polymerase
•RNAP I - rRNAs
•RNAP II - mRNAs
•RNAP III - tRNAs & small ribosomal
RNAs
Different Types of RNA Polymerase

4. (coding strand)
(sense strand)
(non-coding strand)
(anti-sense strand)
RNAP

5. 5’
5’
3’
3’
Template strand
+1
Transcription Initiation Site
Direction of
transcription
“Downstream”
“Upstream”
+2 +3 +4 +5 +6
-3 -1
-2
-4
-5
There is no “zero”

6. •Promoters
- DNA sequences that guide RNAP to the beginning of
a gene (transcription initiation site).
•Terminators
- DNA sequences that specify then termination of RNA
synthesis and release of RNAP from the DNA.
•RNA Polymerase (RNAP)
- Enzyme for synthesis of RNA.
•Steps
• 1) Initiation.
2) Elongation.
3) Termination.
Bacterial (Prokaryotic) Transcription

7. -10 region
RNAP binds a region of DNA from -40 to +20
The sequence of the non-template strand is shown
TTGACA…16-19 bp... TATAAT
“-35” spacer “-10”

8. Important Promoter Features
• The closer the match to the consensus the stronger the
promoter (-10 and -35 boxes)
• The absolute sequence of the spacer region (between
the -10 and -35 boxes) is not important
• The length of the spacer sequence IS important:
TTGACA - spacer (16 to 19 base pairs) - TATAAT
• Spacers that are longer or shorter than the consensus
length make weak promoters

9. Properties of Promoters
• Promoters typically consist of a 40 bp
region on the 5'-side of the transcription
start site
• Two consensus sequence elements:
– The "-35 region", with consensus TTGACA
– The Pribnow box near -10, with consensus
TATAAT - this region is ideal for unwinding.

10. RNA Polymerase Has Many Functions
• Scan DNA and identify promoters
• Bind to promoters
• Initiate transcription
• Elongate the RNA chain
• Terminate transcription
• Be responsive to regulatory proteins
(activators and repressors)
Thus, RNAP is a multisubunit enzyme

11. Transcription in Prokaryotes
• In E.coli, RNA polymerase is a 465 kD complex, with
2 , 1 , 1 ', 1  (holoenzyme).
• Core enzyme is 2 , 1 , 1 ’ (can transcribe but it
can’t find promoters).
•  recognizes promoter sequences on DNA; ' binds
DNA;  binds NTPs and interacts with .
  subunits appear to be essential for assembly and for
activation of enzyme by regulatory proteins.

12.  2 2 2’ = core enzyme
I
 ’
II CORE ENZYME
Sequence-independent,
nonspecific transcription
initiation
+
vegetative
(principal )
70 SIGMA SUBUNIT
interchangeable,
promoter recognition
The assembly pathway of the core enzyme
heat shock
(for emergencies)
32
nitrogen starvation
(for emergencies)
60

13. I
 ’
II
70
RNAP HOLOENZYME -70
Promoter-specific
transcription initiation
In the Holoenzyme:
· ' binds DNA
·  binds NTPs
·  and  ' together make up the active site
·  subunits appear to be essential for assembly and
for activation of enzyme by regulatory proteins. They
also bind DNA.
·  recognizes promoter sequences on DNA

14. Binding of polymerase to Template DNA
• Polymerase binds nonspecifically to DNA with
low affinity and migrates, looking for promoter.
• Sigma subunit recognizes promoter sequence.
• RNA polymerase holoenzyme and promoter
form "closed promoter complex" (DNA not
unwound).
• Polymerase unwinds about 12 base pairs (A-T
rich) to form "open promoter complex“.

15. Finding and binding
the promoter
Closed complex
formation
RNAP bound -40 to
+20
Open complex
formation
RNAP unwinds from -
10 to +2
Binding of 1st NTP
Requires high
purine [NTP]
Addition of next NTPs
Requires lower
purine [NTPs]
Dissociation of sigma
After RNA chain
is 6-10 NTPs long

16. Chain Elongation
Core polymerase - no sigma
• Polymerase is accurate - only about 1 error in
10,000 bases (not as accurate as DNAP III)
• Even this error rate negligible- since many
transcripts are made from each gene
• Elongation rate is 20-50 bases per second -
slower in G/C-rich regions and faster elsewhere
• Topoisomerases precede and follow polymerase
to relieve supercoiling

17. Two mechanisms
1. Rho dependent
Rho - the termination factor protein
An ATP-dependent helicase
– it moves along the RNA transcript, finds the
"bubble", unwinds it and releases the RNA
chain.
Chain Termination

18. Rho-Dependent Transcription Termination
(depends on a protein AND a DNA sequence)
G/C -rich site
RNAP slows down
Rho helicase
catches up
Elongating complex is disrupted

19. The termination function of  factor
The  factor, a hexamer, is a ATPase
and a helicase.

20. Two mechanisms
2) Rho-Independent (Intrinsic)
- termination sites in DNA
– inverted repeat, rich in G:C, which forms a stem-
loop in RNA transcript
– 6-8 nos of A’s in DNA coding for U’s in transcript
Chain Termination

21. Rho-Independent Transcription Termination
(depends on DNA sequence - NOT a protein factor)
Stem-loop structure

22. Rho-independent
transcription
termination
• RNAP pauses when it
reaches a termination site.
• The pause may give the
hairpin structure time to fold
• The fold disrupts important
interactions between the
RNAP and its RNA product
• The U-rich RNA can
dissociate from the template
• The complex is now
disrupted and elongation is
terminated

23. • Bacterial environment changes rapidly.
• Survival depends on ability to adapt.
• Bacteria must express the enzymes required
to survive in that environment.
• Enzyme synthesis is costly (energetically).
• So make enzymes only when required.
Transcription Regulation in Prokaryotes
Why is it necessary?

24. Proteins
Constitutive
• Always expressed
• “housekeeping”
• e.g. glucose metabolizing
enzymes
• Glucose is the preferred
carbon source for
bacteria
Adaptive
• “inducible”
• Made only when needed
• e.g. Lactose metabolizing
enzymes
• Made only if lactose is the
sole carbon source
• Not made if glucose is
present

25. 1. Alternate sigma factor usage: controls selective
transcription of entire sets of genes
s32
s60
vegetative
(principal s)
heat shock
nitrogen
starvation
s70
TTGACA TATAAT
(16-19 bp) (5-9 bp) A
+1
CNCTTGA CCCATNT
(13-15 bp) (5-9 bp) A
+1
CTGGNA TTGCA
(6 bp) (5-9 bp) A
+1
Ways to Regulate Transcription

26. 2. Positive Regulation (activation): a positive regulatory
factor (activator) improves the ability of RNAP to
bind to and initiate transcription at a weak promoter.
Ways to Regulate Transcription
RNAP
-35 -10 +1
Activator
Activator binding site
EXAMPLE: CAP

27. 3. Negative Regulation (repression): a negative regulatory
factor (repressor) blocks the ability of RNAP to
bind to and initiate transcription at a strong promoter.
Ways to Regulate Transcription
RNAP
-35 -10
Repressor
Operator
EXAMPLE: lac REPRESSOR

28. Protein Synthesis is Regulated
Transcriptionally
• Genes that encode proteins with related functions are
grouped into transcriptional units called “operons”
• This ensures that genes for enzymes in the same metabolic
pathway are all made at the same time
Operons have three functional “parts”
1) structural genes: these encode proteins
2) promoter
3) regulatory sequences that interact with regulatory proteins
Sometimes an operon is associated with:
4) regulatory genes: these encode proteins regulating expression
of that operon

29. Structural genes
promoter
Operator (regulatory
sequence that binds a
repressor protein)
Architecture of a typical operon
By regulating a single promoter you can co-ordinate the
expression of three genes (in this example)
RNA transcript covers all genes in the operon
= “polycistronic RNA”

30. Transcription in Eukaryotes

31. RNA polymerases
– Much more complex than prokaryotic RNAP
a) RNAP I – synthesizes ribosomal RNA
b) RNAP II – synthesizes messenger RNA
c) RNAP III – synthesizes transfer RNA and 1 type
of rRNA
Eukaryotic RNAPs have subunits that are
homologous to a, b, and b’ of prokaryotic RNAP;
however, eukaryotic RNAP also contain many
additional subunits.

32. a. Initiation
• Transcription initiation needs promoter
and upstream regulatory regions.
• The cis-acting elements are the specific
sequences on the DNA template that
regulate the transcription of one or
more genes.

33. Eukaryotic promoters
• a) contain a TATA rich region located –25
to -30 from the start of transcription
• b) CCAAT (frequently at –75)
• c) GC box
• d) Some promoters have other sequences
located either upstream or downstream
that maximize the level of transcription
called enhancers

34. structural gene
GCGC CAAT TATA
intron
exon exon
start
CAAT box
GC box
enhancer
cis-acting element
TATA box (Hogness box)
Cis-acting element

35. TATA box

36. • RNA-pol does not bind the promoter
directly.
• RNA-pol II associates with six
transcription factors, TFII A - TFII H.
• The trans-acting factors are the
proteins that recognize and bind
directly or indirectly cis-acting
elements and regulate its activity.
Transcription factors

37. TF for eukaryotic transcription

38. Assembly of RNA pol and transcription
factors at promoter
• Formation of closed complex –TATA binding
protein (TBP) binds to TATA box.
• TBP requires TFIIB
• TFIIA can stabilize TFIIB-TBP complex.
• To TFIIB-TBP complex binds another complex ,
TFIIF and Pol II.
• TFIIF helps target Pol II to promoters..
• TFIIE and TFIIH bind to form the closed complex.
• TFIIH has helicase activity- unwinds DNA

39. Pre-initiation complex (PIC)
RNA pol II
TF II F
TBP TAF
TATA
DNA
TF II
A
TF II
B
TF II E
TF II H

40. • TF II H is of protein kinase activity to
phosphorylate CTD of RNA-pol.
• (CTD is the C-terminal domain of RNA-pol)
• Only the p-RNA-pol can move toward the
downstream, starting the elongation phase.
• Most of the TFs fall off from PIC during the
elongation phase.
Phosphorylation of RNA-pol

41. • The elongation is similar to that of
prokaryotes.
• The transcription and translation do
not take place simultaneously since
they are separated by nuclear
membrane.
b. Elongation

42. • The termination sequence is AATAAA
followed by GT repeats.
• The termination is closely related to
the post-transcriptional modification.
c. Termination