Biochemistry of Medicinals I Phar 6151 CHAPTER THREE
Instructor: Dr. Natalia Tretyakova, Ph.D.
PDB reference correction and design Dr.chem., Ph.D. Aris Kaksis, Associate Professor
RNA Synthesis
I.
Introduction
While DNA is used for information storage, RNA is the means by which this information is articulated in
the organism. It is in essence the vehicle that translates the DNA code into strings of amino acids AA
(i.e. proteins). There are three 3 types of RNA;
• mRNA = Message RNA; a RNA copy of the DNA sequence (gene) used a template for protein synthesis
• tRNA = Transfer RNA; a small RNA 76 bp
that is attached to an amino acid AA which can be added to a growing peptide chain
• rRNA = Ribosomal RNA; component of ribosomes with catalytic and structural function;
involved in protein synthesis; three 3 types exist 23S, 16S, and 5S
DNA and RNA differ in several important ways:
1.) RNA is composed of Uracyl U and not Thymine T
2.) The sugar composition of RNA is composed of ribose and not de-oxy-ribose
3.) Sugar pucker on RNA is 3’ endo and RNA is usually single-stranded
(although it forms hairpins by folding over the same strand)
DNA
RNA
O
O
H H
H
H
N
H
N
Thymine T
N
Uracyl
O
N
H
H
H
H
H
O
H
U
O
O
H
O
H
Base
Base
O
2'-de-oxy-ribose
H
D-ribose
H
O
H
O
O
Base
O
O
O
H
H
H
O
H
Base
O
H
O
H
O
H
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Quantitiy of RNA in the bacteria E. coli
Type
rRNA
Relative amount, %
tRNA
mRNA
II.
B.
80
15
5
Sed. Coeff. Sv
23
16
5
4
Mass, kD
1.2•103
0.6•103
3.6•101
2.5•101
heterogeneous
# of Nucleotides
3700
1700
120
75
RNA SYNTHESIS IN PROKARYOTES
RNA Polymerases
The synthesis of RNA is catalyzed by RNA polymerases. They function by catalyzing the
step-by-step addition of ribonucleotides to the 3' end of a RNA polymer chain.
These enzymes are important for the production of mRNA, tRNA and rRNA from a DNA template.
1. POLYMERIZATION REACTION
Like all polymerization reactions, the RNA synthesis is characterized by three 3 stages;
initiation, elongation, and termination.
RNA polymerization requires:
•
•
•
•
Double stranded DNA template strand; rarely does ssDNA serve as template
Does not need a primer: just GTP or ATP
Ribonucleoside 5'-triphosphates, ATP, GTP, UTP and CTP (NTPs)
Mg2+ to activate the NTPs
RNAn bases + NTP
RNAn+1 + O-O2P-O-PO-2O
RNA polymerase does the following;
• Searches DNA and finds initiation (promotor) sites
• Unwinds a short stretch of double-helical DNA to produce a ssDNA template
• Adds one 1 NTP after another by the nucleophilic attack of the 3'- hydroxyl -O on the alpha
 phosphate of the NTP; is totally processive; one enzyme makes the entire transcript (mRNA).
• Addition of NTPs follows Watson-Crick base pairing between the DNA template and the
incoming NTPs;
• Unlike DNA Polymerases, RNA Polymerases do not have nuclease activity;
not able to proof read for mismatches.
• Detects termination signals

• Activator and repressor proteins modulate the rate v of transcription by RNA Pol.
The sequence of the newly synthesized RNA is complementary to the sequence of its
template DNA (antisense strand) and is identical to the sequence of the coding (sense) strand
(with the exception of Us instead of Ts).
5' … A T G G C C T G G A C T T C A… 3' Sense strand of DNA
3' … T A C C G G A C C T G A A G T… 5' Anti-sense strand of DNA
 Transcription of anti-sense strand
5' … A U G G C C U G G A C U U C A… 3' mRNA
 Translation of mRNA
Met-Ala-Trp-Thr-Ser- Peptide
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2.
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RNA Polymerase Structure
RNA polymerases from bacteria are very large (450kD) and are composed of four 4 kind of subunits:
2’(holoenzyme).
Subunit


'
holoenzyme
B.
Number Mass kD
Function
2
37 Binds regulatory sequences
1
151 Bonds NTPs, forms phosphodiester bonds
1
155 Binds DNA template
1
70 Recognizes promoter and initiates synthesis
RNA Replication
Transciption or RNA synthesis occurs along one of the strands of DNA and proceeds 5’3’.
The DNA strands are locally unwound and separated (replication bubble, about 1.6 turns of B-helix).
1.
Initiation
DNA sequences called promoters determine where transcription begins. RNA polymerase will bind to
these sites in the absence of ribo-nucleoside tri-phosphates. Common motifs have been observed for these
sequences:
Consensus Prokaryotic Promoter Sequence
-35
-10
+1
5'———TTGACA———————TATAAT————••••••••••••••••••••••••••••••••••••3'

Promoter sequence
Start site  Coding sequence

•
The efficiency of the promoter is measured as the amount of transcription per unit time. The more
transcripts made the better the promoter. The best promoters are ones that are as close to the consensus
sequence as possible. Strong promoters result in frequent transcription initiation (e.g. 2/sec). Mutations in
the promoter region can severely limit a promoters function.
• ’ (holoenzyme) is necessary for initiation. The 5 subunit is necessary for increasing the specificity for
the promoter 10 000 fold over a random DNA sequence.
- Promoter sequence found by sliding of the holoenzyme along DNA duplex until it finds the right
H-bond motif. This takes place without unwinding the double helix.

- Rate of v = 1010 M-1s-1 for binding to promoter sequence.
- Different subunits for different promoters (i.e., heat shock)
• Before RNA synthesis can begin, RNA Polymerase unwinds a 17 bp reading area of duplex DNA by 1.6
turns to form open promoter complex. This imparts positive (+) DNA supercoiling that can be relieved by
topo-isomerases. Negative (-) supercoiling of DNA can promoter efficiency by facilitating DNA
unwinding.
• RNA polymerase needs no primer, however, a molecule of GTP or ATP is used to start transcription.
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Mechanism of Nucleotide Addition
G or A
O
O
O
O P O P O P O
O
O
5'
O
O
3'
Strand
3'
O
O H
OH
O
O
O P O P O P O
O
O
O
2+
Mg
2.
Template
Base
O
5'
O H
Elongation
Elongation after the addition of the first base to GTP or ATP proceeds quickly until termination signal is
encountered.
•
•
•
•
The holoenzyme, ’ loses the  subunit
A transcription bubble is formed
Contains a RNA-DNA duplex of 12 bp
the RNA Pol locally melts the DNA duplex followed by rewinding
(the transcription bubble does not increase  in size)
•
•
Rate of v = 50 NTP's per sec.
Mistakes 1 in 10 000 -100 000 (no proofreading since no nuclease activity)

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RNA Polymerization Reaction
Page 5
5'
3'
3'
5'

RNA
Polymerase
3'
5'
5'
3'
 RNA Polymerase
17 bp  unwound
  NTP's
Coding
strand
Rewinding 
5'
3'
3'
5'
5'ppp 
3.
RNA-DNA  12 kb Template strand
Termination
During the last phase of synthesis, the polymerization reaction is stopped by a specific signal, the RNA-DNA
complex comes apart, the melted DNA rewinds, and the RNA polymerase dissociates from the DNA. There are
two 2 mechanism for termination.
1.) Formation of a stable hairpin from palindromic GC rich-region followed by AT-rich region. The
enzyme pauses after this and the RNA dissociates from the template and enzyme complex.
/-C-\
U G
|
|
UG
\
/
G•C
A• U
C•G
C•G
G•C
C•G
C•G
G•C
5'- U•C•C•C•A•G A• U • U • U -3'
RNA Hairpin
2.)
The Rho () protein factor is a hexamer of identical 419-residue subunits. It recognizes certain
regions of RNA that come before the termination site and wraps around them in an ATP driven 
process. This them allows the RNA to be pried off of the RNA-DNA duplex.
III.
RNA Synthesis in Eukaryotes
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Unlike prokaryotes, eukaryotes transcription and translation takes place in different compartments.
Consequently, control of this processes is much more  elaborate. There are three 3 big differences between
prokaryotic and eukaryotic transcription.
1.)
2.)
3.)
A.
RNA requires a 5' CAP
RNA requires a 3' poly (A) tail.
RNA is extensively spliced to remove introns  off ? RNA.
RNA POLYMERASES
The synthesis of RNA is catalyzed by three 3 RNA polymerases that all reside in the nucleus. They function
by catalyzing the step-by-step addition of ribo-nucleotides to the 3' end of a
RNA polymer chain. These enzymes are important for the production of mRNA, tRNA and rRNA from a
DNA template.
Type Localization ? RNA produced
Inhibition by amanitin
I
II
III
Nucleolus
Nucleoplasm
Nucleoplasm
rRNA
mRNA
tRNA,rRNA
Insensitive
Strongly inhibits
Weakly inhibits
Amanitin = Octapeptide derived from the Death Cap mushroom
•
RNA Pol II is responsible for transcription to mRNA
-
8-12 subunits
Two 2 subunits ( 220 kD and 140 kD) responsible for synthesis
Regulated by phosphorylation of C-carboxyl-terminal domain (CTD)
Subunits 5, 6, and 8 are in Type I and II.
• Same chemical rules that apply for the polymerization reaction for prokaryote synthesis apply to
eukaryote
synthesis
Biochemistry of Medicinals I
B.
RNA REPLICATION
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There are many similarities to prokaryotic transcription, but there are also many important differences.
1.
Initiation
Eukaryote promoters are also located upstream  from the coding sequence for the transcript.
Consensus Eukaryotic Promoter Sequence
-110
-40
-25
+1
5'———CAAT box——————GC box———TATA box———••••••••••••••••••••••••••••••3'

Promoter sequence
Start site 
Coding sequence
•
•
•
•
TATA box is necessary but not sufficient
CAAT box and GC box are present but not always
Synthesis begins with GTP or ATP
5' end is quickly modified to a CAP form with GTP
H
H
Caps 0,1,2
O
O
P
O

O
H
O
+
H
N
N
O
H O
H
N
N
N
H
O
O
O
5' End Capped
H
H
P
O
P
O
H
O
A or
G
O
H
O H
H
O
O
O
P
O
H
Caps 1 and 2
H
O
H
H
Base
O
O
H
O
H
H
Cap 2
DNA 3' Transcription and 5' capping
Figure . Formation of the primary
transcript and its processing during
maturation of mRNA in a eukaryotic
cell .
The 5' cap (in red) is added before
synthesis of the primary transcript
is complete.
Primary transcript Completion of  primary transcript RNA polymerase
5'
3'
5'  Cap  Exon  Intron

Noncoding end sequence 
Cleavage, poly-adenylation  and splicing + +
mature RNA
-AAA(A)n 3'
A non coding sequence following
the last exon is shown in orange.
S plicing can occur either before or
after the cleavage and
poly-adenylation steps.
All of the processes shown here
take place within the nucleus.
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Transcription Factors
RNA polymerase II is unable to initiate synthesis by itself, it must be guided to the start site by a
transcription factor (TFII). This is large family of proteins that interact with RNA Pol II in order to facilitate
polymerization.
They bind to the start site in the following order
1.)
2.)
3.)
4.)
5.)
TFIID; contains TATA box binding protein
TFIIA
TFIIB; binds over start site for coding region
RNA Pol II
TFIIE
Table 26-1. Proteins Required for Transcription at the RNA Polymerase II Promoters of
Eukaryotes
Number of
Transcription factor subunits Subunit MW
Functions
Initiation
RNA polymerase II
12 10 000-220 000 Catalyzes RNA synthesis
TBP (TATA-binding protein) 1
38 000 Specifically recognizes the TATA box
TFIIA
3 12 000,19 000, Stabilizes binding of TFIIB and TBP to the promoter
35 000
TFIIB
1
35 000 Binds to TBP; recruits RNA polymerase-TFIIF complex
TFIID
12 15 000-250 000 Interacts with positive and negative regulatory proteins
TFIIE
2 34 000, 57 000 Recruits TFIIH; ATPase and helicase activities
TFIIF
2 30 000, 74 000 Binds tightly to RNA polymerase II; binds to TFIIB and
prevents binding of RNA polymerase to
nonspecific DNA sequences
TFIIH
12 35 000-89 000 Unwinds DNA at promoter; phosphorylates RNA
polymerase; recruits nucleotide-excision repair
complex
Elongation *
1
80 000
ELL‡
P-TEFb
2 43 000,124 000
SII (TFIIS)
1
38 000
Elongin (SIII)
3 15 000, 18 000,
110 000
* All Elongation factors suppress the pausing or arrest of transcription by the
RNA polymerase II-TFIIF complex.
‡
The name is derived from the term eleven-nineteen lysine-rich leukemia. The gene for the factor ELL is
the site of chromosomal recombination events frequently associated with
the cancerous condition known as acute myeloid leukemia.
Biochemistry of Medicinals I
5'
-30 TATA
+1 Inr
3'
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3'
RNA Polymerase II Complex
5'
 TBP (or TFIID and/or TFIIA)  TFIIB  TFIIE  TFIIH
 TFIIB - RNA polymerase II 


 polymerase
Figure .
Transcription at RNA
 RNA
II
TFIIF
polymerase II
TFIID
 release and
RNA polymerase II
promoters.
 de-phosTFIIA TFIIB
(a) The sequential
 -phorylation
TFIIF
assembly of a complex
TBP

TFIIH
consisting of TBP

(often with TFIIA),



 Closed complex
TFIIB plus

 DNA unwinding to produce open complex
RNA polymerase II,

TFIIE, TFIIF, and TFIIH

results in a
 RNA

closed complex.

TBP often binds as part
 Termination
of a larger complex

called TFIID.

Some of the TFIID


Unwound DNA 

Elongation
subunits play a role in
 Phosphorylation of RNA polymerase II,
Elongation 

transcription regulation
 initiation, and release of elongation complex
factors   
(see Fig. 28-32).
 TFIIH
The DNA is unwound at
 
the Inr region by the
 TFIIE
Inr
helicase activities of

P P
TFIIH and perhaps
P P
TFIIE, creating an
P P
open complex.
The carboxyl-terminal domain of the largest RNA
polymerase II subunit is phosphorylated by TFIIH,
and the polymerase then escapes the promoter and
begins transcription.
Elongation is accompanied by the release of many
transcription factors and is also enhanced by
elongation factors (see Table 26-1).
After termination, the RNA polymerase is released,
de-phosphorylated, and recycled.
(b) The structure of TBP (gray) bound
to DNA (blue and white).
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Other factors may also be necessary depending on the site such as special
transcription factors and enhancers.
•
•
•
•
•
Page 10
Enhancers are able to stimulate transcription thousands 1000÷3000 of bp away.
Enhancers can be upstream , downsteam  and even in the sequence
They are generally cell specific.
Ex. Enhancer that binds glucocorticoid steroids.
Virus can also have enhancers that are tissue and species specific
2. Termination
Termination of transcription is not well understood. However, all transcripts end up with a poly (A) tail,
which appears to be important for efficient translation.
•
•
•
An endonuclease recognizes and cleaves the AAU / AA sequence
A poly (A) polymerase adds 250 A's to the end.
The transcript is transported out of the nucleus