Rna polymerase

This PPT describes structure, types, evolutionary changes and function of prokaryotic and eukaryotic RNA polymerases.

1. RNA Polymerase
RNAP
Ali Raza
School of Life Sciences, University of Science and Technology
of China

2. Definition
•In all organisms, RNA synthesis is carried out by Enzymes --known as RNA
polymerases (RNAPs) --that transcribe the genetic information from DNA in a
highly-regulated, multi-stage process.
•RNAP is the key enzyme involved in creating an equivalent complementary
RNA copy of a sequence of DNA.
•Atranscription factorand its associated transcriptionmediator complexmust
be attached to aDNA binding sitecalled apromoter regionbefore RNAP can
initiate the DNA unwinding at that position.

3. Complex Depiction of Central Dogma

4. Evolutionaryship of general transcription
factors

5. Cellular RNA polymerases in all living organismsare evolutionary related
LUCA-Last Universal Common Ancestor

6. Schematic representation of the evolution of the main cellular RNA polymerases. The five subunits present in eubacteria are
coloured light blue, whereas the seven subunits that were added to form the ancestral core RNA polymerase are coloured dark
blue. The other subunits present in some enzymes are coloured green, redand yellow, as indicated. The phylogeny depicted,
with eubacteria being the sister group of Archaea and eukaryotes and with RNAPI being the sister group of RNAPII and III, is
generally accepted as being most likely. The different branch lengths of RNAPI, II and III also represent their relative amino acid
substitution rates.

8. The efficiency of E. coliRNA polymerase is around 40 nt/sec at 37ºC, and
requires Mg
2+
(RNA polymerase of T3 and T7 are single polypeptides with a efficiency of
200 nt/sec)
The enzyme has a non-spherical structure with a projection flanking a
cylindrical channel
The size of the channel suggests that it can bind directly to 16 bp of DNA
The enzyme binds over a region of DNA covering around 60 bp
E. coli RNA polymerase

10. αsubunit: 36.5 kDa, encoded by rpoAgene. Required for core protein
assembly, and also play a role in promoter recognition. Assembly of β
andβ’.
βsubunit: 151 kDa, encoded by rpoBgene. DNA-binding active
center.
β’ subunit: 155 kDa, encoded by rpoCgene. Responsible for binding
to the template DNA. Uses 2 Mg
2+
ions for catalytic function of the
enzyme.
subunit: 91 kDa, encoded by rpoZgene. Restores denatured RNA
polymerase to its functional form in vitro. It has been observed to offer
a protective/chaperone function to the β' subunit in Mycobacterium
smegmatis.
RNA Polymerase subunits

11. Three RNA polymerases; many with subunits
pol I-only the large ribosomal RNA subunit precursors (18, 5.8, 28S )
pol II-all pre-mRNAs, some small nuclear RNAs
(snRNAs), most small nucleolar RNAs (snoRNAs)
used in rRNA processing
pol III -tRNAs,, U6 snRNA, 7SL RNA (in SRP),
and other s5S rRNAmall functional RNAs
pol IVand V-plants, siRNA andheterochromatinin the nucleus
Mitochondria and Chloroplasts
-combination of phage-like (single subunit) and
bacterial-like (multi-subunit)
Eukaryotic viruses
-can take over host RNAP or encode own in some large viruses (e.g. vaccinia)
EukaryoticRNA Polymerase Types
•RNA Pol I transcribe 1 gene at
~200 copies. The gene for the
45S pre-rRNA is present in
tandem array.
•RNA Pol II transcribe ~25,000
genes
•RNA Pol III transcribe 30-50
genes at variable copy numbers.

12. Subunit composition of eukaryotic RNA polymerases
•All three yeast polymerases have five core
subunits that exhibit some homology with the α,
β, β’ andsubunits in E. coliRNA polymerase.
•RNA polymerases I and III contain the same two
non-identical a-like subunits, whereas polymerase
II has two copies of a different a-like subunit.
•All three polymerases share four other common
subunits. In addition, each RNA polymerase
contains three to seven unique smaller subunits.
•The largest subunit (1) of RNA polymerase II also
contains an essential C-terminal domain (CTD).
27 (yeast) to 52 (human) copies of (YSPTSPS).
•Phosphorylation of CTD is important for
transcription and RNA processing.

13. Comparison of 3-D structures of bacterial and eukaryotic RNA polymerases
(subunits 4
and 7 are
missing)
RNA polymerase II from yeast has been extensively characterized
Contains two large subunits that are homologs of prokaryotic RNA polymerase subunits band b’
Contains 10 smaller subunits, including homologs of aand r
Structural features
-Thumb
-DNA-binding channel
-Channel for single-stranded RNA

14. Transcription Cycle

15. Each gene has three regions
1.5’Promoter, attracts RNA polymerase
-10 bp 5’-TATAAT-3’
-35 bp 5’-TTGACA-3’
2.Transcribed sequence (transcript) or RNA coding sequence
3.3’Terminator, signals the stop point
Prokaryotic Transcription
Bases preceding
this are numbered
in a negative
direction
There is no base
numbered 0
Bases to the right are
numbered in a
positive direction
Sometimes termed
the Pribnowbox,
after its discoverer
Sequence elements
that play a key role in
transcription

16. sigma subunit positions RNA
polymerase for correct initiation.
Upon initiation of transcription,
sigma subunit dissociates.
Transcription Initiation in Prokaryotes

17. Elongation
RNA polymerase adds ribonucleotides in 5’to 3’direction.
RNA polymerase catalyzes the following reaction:
NTP + (NMP)n (NMP)n+1+ PPi
DNA
Mg++
RNA polymerase

18. (1)Intrinsic
Termination site on template
DNA consists of GC-rich
sequences followed by 6-8 A’s.
Intra-molecular hydrogen
bonding causes formation of
hairpin loop.
Termination
In E. coli,this structure signals
release of RNA polymerase, thus
terminating transcription.

19. Termination of transcription occurs beyond the coding
sequence of a gene. This region is 3’untranslated region (3’
UTR), which is recognized by RNA polymerase.

20. (2) Rho factor (hexameric
protein) dependent:
These termination signals do
not produce hairpin loops. rho
binds to RNA at
rut(rhoutilisation site)or
rho pulls RNA away from RNA
polymerase.
72 nucleotide stretch
C-rich/G-poor
Lack secondary structure
rutsite

21. Eukaryotic Transcription

23. Basal (‘General’) Transcription Factors for RNA Polymerase II
Total: 43-44
polypeptides
and over 2
million Daltons.

24. The association of TBP with TAFIs,TAFIIs, TAFIIIs, and PTF/SNAPc directs TBP to
different promoter classes. The distribution of TBP among these factors
contributes to the global regulation of gene expression. From Lee and Young
(1998) Regulation of gene expression by TBP-associatedproteins. Genes Dev.
12, 1398.
Four complexes function as class-specific promoter selectivity factors

25. Sequences that can act as promoters(TATA is preferred)
Promoter

26. •Regulatory Elements/Response Element -Response elementsare the
recognition sites of certain transcription factors Most of them are located
within 1 kb from the transcriptional start site.
•Enhancer elements -upon binding with transcription factors
(activators), can enhance transcription; located either upstream or
downstream of the transcriptional initiation site.
•Upstream enhancer elements
•Downstream enhancers
•Distal enhancer elements
•Silencers -upon binding with transcription factors (repressors), can repress
transcription.
Other Regulatory Elements

27. Pre-Initiation Complex Formation
The closed complex of transcription (eukaryotic) is formed in the
following steps:
1.The TATA-binding protein (TBP) binds to the TATA box.
2.TPB is bound by TFIIB, which also binds to the DNA on either
side of the TBP.
3.The TFIIB-TBP complex is bound by another complex consisting
of TFIIF and RNA pol II.
4.TFIIE and H bind to complete the closed complex.
5.TFIIH has a helicase activity that can unwind the DNA around
the transcription start site (+1).

28. Recognizes and binds to TATA box; TBP + 10
TBP associated factors; position set
RNA Pol II bound to DNA and
general transcription factors
TBP bends DNA ~80
o
and forces
open the minor groove.

29. Recognizes and binds to TATA box; TBP + 10
TBP associated factors
Binds and stabilizes the TFIID complex
Recruits RNA pol II + TFIIF to the location
Two subunits -RAP38 & RAP74. Rap74 has a
helicase activity; RAP38 binds RNAPolII
Two subunits -recruits TFIIH to the complex
thereby priming the initiation complex for
promoter clearance and elongation
complex of 9 subunits. One w/ kinase activity;
one w/ helicase activity; one is a cyclin (cdk7)

30. General transcription factor structures.
(a)Structure of TBP (green) bound to TATA-
DNA with B-form DNA (greyand red)
modeled upstream and downstream of the
TATA box.
(b)Structure model of the TBP-TFIIA-TFIIB-
DNA complex. TBP (green) is shown from
the top binding to the TFIIB core domain
(TFIIBc, blue) and TFIIA (large subunit
magenta, small subunit yellow). The zinc
ribbon domain (shown as β strands with
red Zn atom) connects to the B-finger
domain is normally located in the PIC
within the Pol II active site and is
connected to the TFIIBcdomain through a
flexible linker.
This model is a composite of the DNA-TBP-
TFIIBc, DNA-TBP-TFIIA, and the TFIIB Zn
ribbon NMR and crystal structures.

31. EM structure of the Pol II-Mediator complex. Mediator (dark blue) shown with head, middle, and
tail domains. Pol II (gold) shown with modeled upstream and downstream DNA (orange). The dot
represents the presumed beginning of the CTD.
Mediator
Complex needed for a response to
transcriptional activators by purified RNA
Pol II plus GTFs
Yeast Mediator has 20 subunits, including
Srb2, 4, 5, 6; Srb7, Rgr1, Gal11, Med1, 2, 6,
7, Pgd1, Nut1, 2, and others
RNA Pol II + Mediator(+ some GTFs) =
Holoenzyme

32. Transcription initiation in the cell often requires the local recruitment of chromatin-
modifying enzymes, including chromatin remodeling complexes and histone
acetylases -greater accessibility to the DNA present in chromatin

33. Distinct forms of RNA polymerase used for
initiation and elongation: RNA Pol II
Model: Phosphorylation of Pol IIa to make Pol IIo is
needed to release the polymerase from the initiation
complex and allow it to start elongation.
CTD has repeat of (YSPTSPT)
26-50.
CTD = C-terminal domain

34. RNAP making short
RNAs, its stalled at
+10 -+12.
TFIIH causes further
DNA unwinding,
allowing the bubble to
grow and RNAP to go
to elongation phase.
Polymerization of 1st few NTPs and
phosphorylation of CTD leads to promoter
clearance. TFIIB, TFIIE and TFIIH dissociate,
PolII+IIF elongates, and TFIID + TFIIA stays at
TATA.

35. Stages in Initiation of Transcription
Bacterial transcription
•Closed complex:
holoenzyme+promoter
•Open complex (DNA
melting, notneed ATP)
•Abortive transcription
•Productive initiation
–Transcribe past +9
–Sigma dissociates
•Elongation
Eukaryotic transcription
•Preinitiation complex (PIC)
assembly
•PIC activation (DNA melting,
needs ATP)
•Abortive transcription
•Productive initiation
–CTD phosphorylated
–Promoter clearance
•Elongation

36. mRNA Differences Between Prokaryotes And Eukaryotes
Prokaryotes
1.mRNA transcript is mature, and used directly for translation without modification.
2.Since prokaryotes lack a nucleus, mRNA also is translated on ribosomes before it is
transcribed completely (i.e., transcription and translation are coupled).
3.Prokaryote mRNAs are polycistronic, they contain amino acid coding information for
more than one gene.
Eukaryotes
1.mRNA transcript is not mature (pre-mRNA); must be processed.
2.Transcription and translation are not coupled (mRNA must first be exported to the
cytoplasm before translation occurs).
3.Eukaryote mRNAs are monocistronic, they contain amino acid sequences for just one
gene.

37. Facts To Remember
DNA-dependent RNA synthesis:
1.Starts at a promotersequence, ends at termination signal
2.The first 5’-triphosphate is NOT cleaved
3.Proceeds in 5’ to 3’direction
4.New residues are added to the 3’ OH
5.The template is copied in the 3’ –5’direction
6.Forms a temporary DNA:RNA hybrid
7.Transcription rate ( 50 to 90 nts/sec)
8.RNA polymerase has complete processivity
9.RNA polymerase adds ribonucleotides (rNTPs) not deoxynucleotides (dNTPs)
10.RNA polymerase does not have the ability to proofread what they transcribe
11.RNA polymerase can work without a primer
12.RNA will have an error 1 in every 10,000 nt (DNA is 1 in 10,000,000 nt)