r r
T ansc ipton
Md . Ab u Rakib d r
r r
P ofesso
r d M r
Dept. of Biochemist y an olecula Biology
r
Unive sity of Rajshahi
r d d RNA, like DNA, is a polyme consistng of nucleot es joine
r d r d r r r
togethe by phospho ieste bon s. Howeve , the e a e
r r d r r r d
seve al impo tant ife ences in the st uctu es of DNA an RNA.
r r
St uctu e of RNA
r r
St uctu e of RNA
r r d d r r r RNA has a p ima y an a secon a y st uctu e.
Classes of RNA
Classes of RNA
r r r
T ansc ipton is the synthesis of an RNA molecule f om a DNA template.
r r r
T ansc ipton is the synthesis of an RNA molecule f om a DNA template.
r r d r r r
All cellula RNAs a e synthesize f om DNA templates th ough the p ocess
r r
of t ansc ipton.
r d r In eplicaton, all the nucleot es in the DNA template a e
d r r r
copie , but, in t ansc ipton, only small pa ts of the DNA
r r
molecule—usually a single gene o , at most, a few genes—a e
r r d
t ansc ibe into RNA.
r d r d d r
Because not all gene p o ucts a e nee e at the same tme o
r r
in the same cell, the constant t ansc ipton of all of a cell’s d
genes woul be highly inefcient.
r r r d d
Fu the mo e, much of the DNA oes not enco e a functonal
r d d r r d
p o uct, an t ansc ipton of such sequences woul be pointless.
r r r d d
T ansc ipton is, in fact, a highly selectve p ocess: in ivi ual
r r r d r r d r d d
genes a e t ansc ibe only as thei p o ucts a e nee e .
r r r d
Compa ison of T ansc ipton an Replicaton
r r r r r
T ansc ipton is in many ways simila to the p ocess of eplicaton.
r r r r r r
T ansc ipton equi es th ee majo components:
1. A DNA template;
r r r d d d d
2. The aw mate ials (subst ates) nee e to buil a new RNA molecule; an
r r r r r
3. The t ansc ipton appa atus, consistng of the p oteins necessa y to catalyze the
synthesis of RNA.
r r r
Requi ements of t ansc ipton
r r r d
The template fo RNA synthesis, as fo DNA synthesis, is a single st an of the DNA double helix.
r r r
Unlike eplicaton, the t ansc ipton of a gene takes place on only one of the two
d r d r
nucleot e st an s of DNA (Figu e 13.4). The nucleot e st an use fo t ansc iptond r d d r r r
r d r d r r d d r d
is te me the template st an . The othe st an , calle the nontemplate st an , is not
rd r r r d d r d
o ina ily t ansc ibe . Thus, within a gene, only one of the nucleot e st an s is
r r r d r r r
no mally t ansc ibe into RNA (the e a e some exceptons to this ule).
r r d r d
The t ansc ibe st an
r r d r r
A t ansc ipton unit inclu es th ee c itcal regions:
- a p omote r r
- an RNA-co ing sequence, and d - a te minato . r r
r r
The p omote is a DNA sequence that the
r r r r d d
t ansc ipton appa atus ecognizes an bin s. It
d r d
in icates which of the two DNA st an s is to be r d ea as the template an the i ecton ofd d r
r r r r d r
t ansc ipton. The p omote also ete mines
r r r r d
the t ansc ipton sta t site, the f st nucleot e
r r d
that will be t ansc ibe into RNA. In most
r r r r d
t ansc ipton units, the p omote is locate
r r r
next to the t ansc ipton sta t site but is not,
r r d
itself, t ansc ibe .
d r
RNA-co ing egion, a
sequence of DNA
d d
nucleot es that is copie into an RNA molecule.
rd
The thi component of the
r r
t ansc ipton unit is the
r r
te minato , a sequence of
d r
nucleot es that signals whe e
r r d
t ansc ipton is to en .
r r r r
Te minato s a e usually pa t of d
the RNA-co ing sequence; that
r r r
is, t ansc ipton stops only afe
r r d
the te minato has been copie into RNA.
r r r d d r r
A t ansc ipton unit is a st etch of DNA that enco es an RNA molecule an the sequences necessa y fo its
r r
t ansc ipton.
r r
The t ansc ipton unit
r r r r
The Subst ate fo T ansc ipton
d r r d r
RNA is synthesize f om ibonucleosi e t iphosphates
r r d r
( N T Ps ; Fig u e 1 3 .7 ). In s y n th e s is , n u c le ot e s a e
dd d r r
a e one at a tme to the 3 -OH g oup of the g owing′
r r d r
RNA molecule. Two phosphate g oups a e cleave f om the
r d r r
incoming ibonucleosi e t iphosphate; the emaining
r r d r d
phosphate g oup pa tcipates in a phospho ieste bon d r
that connects the nucleot e to the g owing RNA molecule.
r r r dd
The ove all chemical eacton fo the a iton of each d
nucleot e is:
r
RNAn + NTP → RNAn+1 + PPi
r r r r d r
whe ePPi ep esentspy ophosphate. Nucleot i esa e
dd d d d
always a e to the 3 en of the RNA molecule, an the′ d ri ecton of t ansc ipton is the efo e 5 →3 . r r r r ′ ′
r r r d d Bacte ial RNA polyme ases a e fve subunits (in ivi ual
d r
polypept e chains) that make up the co e enzyme: two copies
d d
of a subunit calle alpha (α) an single copies of subunits beta
r d r
(β ), b e ta p im e (β ), a n om e g a (ω ) (Fig u e 1 3 .9 ).′
r r r
The ω subunit is not essental fo t ansc ipton, but it helps r
stabilize the enzyme. The co e enzyme catalyzes the elongaton
dd d r
of the RNA molecule by the a iton of RNA nucleot es. Othe
d r r r
functonal subunits join an leave the co e enzyme at pa tcula
r r r
stages of the t ansc ipton p ocess.
r r d r
The sigma (σ) facto cont ols the bin ing of RNA polyme ase to
r r r
the p omote . Without sigma, RNA polyme ase will initate
r r r d r
t ansc ipton at a an om point along the DNA. Afe sigma has
d r r
associate with the co e enzyme (fo ming a holoenzyme), RNA
r d r r r d
polyme ase bin s stably only to the p omote egion an
r r r r r
initates t ansc ipton at the p ope sta t site.
r r r Bacte ial p omote s
r r r r r r r r r d r d
Essental info maton fo the t ansc ipton unit—whe e it will sta t t ansc ibing, which st an is to be ea ,
d d r r dd d d
an in what i ecton the RNA polyme ase will move—is imbe e in the nucleot e sequence of the
r r
p omote .
r r r r r d r r r d r
P om ote s a e D NA s e q u e n c e s th a t a e e c og n iz e b y th e t a n s c ip ton a p p a a tu s a n a e requi e fo t ansc ipton to take place. In bacte ial cells, p omote s a e usually a jacent to an RNA-co ingr d r r r r r r r d d sequence.
Most of the nucleot es within the p omote s va y in sequence, sho t st etches of nucleot es a e common d r r r r r d r
r r r d d r r r r
to many. Fu the mo e, the spacing an locaton of these nucleot es elatve to the t ansc ipton sta t site
r r r r r r d r d
a e simila in most p omote s. These sho t st etches of common nucleot es a e calle consensus
sequences; “consensus sequence” efe s to sequences that possess consi e able simila ity, o consensus r r d r r r
r r d
(Fig u e 1 3 .1 0 ). T h e p e s e n c e of c on s e n s u s in a s e t of n u c le ot e s u s u a lly im p lie s th a t th e
d r
sequence is associate with an impo tant functon.
r d d r r r
The most commonly encounte e consensus sequence, foun in almost all bacte ial p omote s, is
r d r d
cente e about 10 bp upsteam of the sta t site. Calle the –10 consensus sequence o , sometmes, ther
r
P ibnow box, its consensus sequence is TATAAT.
r r r r
Anothe consensus sequence common to most bacte ial p omote s is TTGACA, which
r d r r d r d
lies app oximately 35 nucleot es upst eam of the sta t site an is te me the −35 consensus sequence (s e e Fig u e 1 3 .1 1 )r
r r d d r r d r
A p omote is a DNA sequence that is a jacent to a gene an equi e fo
r r r r r r r
t ansc ipton. P omote s contain sho t consensus sequences that a e impo tant
r r
in the initaton of t ansc ipton.
r r r Rifamycins a e a g oup of antbiotcs that kill bacte ial cells by
r r d d
inhibitng RNA polyme ase. These antbiotcs a e wi ely use to
r r d
t eat tube culosis, a isease that kills almost 2 million people
r d d r r r r d r
wo l wi eeachyea . Thest uctu esof bacte ial an euka yot ic
r r d r r
RNA polyme ases a e sufciently ife ent that ifamycins
r r r rr
inhibit bacte ial RNA polyme ases without inte fe ing with
r r r r
euka yotc RNA polyme ases. Recent esea ch has
demonst ate that seve al ifamycins inibit RNA polyme ase byr d r r r
d r r
bin ing to the pa t of the RNA polyme ase that clamps on to
d r r r
DNA an jamming it, thus p eventng the RNA polyme ase f om
r r r
inte actng with the p omote on the DNA.
r r r r
The P ocess of Bacte ial T ansc ipton consists of Initaton, Elongaton, d r
an Te minaton
r r d d d r
T ansc ipton can be conveniently ivi e into th ee stages:
1. Initaton, in which the t ansc ipton appa atus assembles on the r r r
r r d
p omote an begins the synthesis of RNA;
2. Elongaton, in which DNA is th ea e th ough RNA polyme ase, the r d d r r
r d d dd d
polyme ase unwin ing the DNA an a ing new nucleot es, one at a
d r r d d
tme, to the 3 en of the g owing RNA st an ; an′
3 . Te minatonr , the ecogniton of the en of the t ansc ipton unit an r d r r d
r r
the sepa aton of the RNA molecule f om the DNA template.
r
Bacte ial cells possess two majo types of te minato sr r r .
d d r r
Rho- epen ent te minato s a e able to cause the te minaton of t ansc ipton only in the p esence of anr r r r r
r r d r r
ancilla y p otein calle the ho facto .
d d r r r r r
Rho-in epen ent te minato s (also known as int insic te minato s) r a e able to
d r r r
cause the en of t ansc ipton in the absence of ho.
d d r r d d r r
Rho-in epen ent te minato s Rho-in epen ent te minato s, which make up about 50
r r r r r
pe cent of all te minato s in p oka yotes, have two common featu esr . r
Fi st, they contain inve ter d repeats (sequences of nucleot es on one st an that a e d r d r
r d d r r d r r r d
inve te an complementa y). When inve te epeats have been t ansc ibe into RNA,
r d r r r r r
a hai pin secon a y st uctu e fo ms (Figu e 13.13).
d
Secon , in ho in epen ent te minato s, a st ing of seven to nine a enine nucleot es r d d r r r d d
d r d r r r r r d
follows the secon inve te epeat in the template DNA. Thei t ansc ipton p o uces
r r d r r r r d
a st ing of u acil nucleot es afe the hai pin in the t ansc ibe RNA.
r r r r
The st ing of u acils in the RNA molecule causes the RNA polyme ase to pause, allowing tme fo
r r r r r d r
the hai pin st uctu e to fo m. The hai pin establizes the DNA–RNA pai ing, causing the RNA
r r
molecule to sepa ate f om its DNA template.
r d d r r r r
Sepa aton may be facilitate by the a enine–u acil base pai ings, which a e elatvely weak
r d r r r r r d r
compa e with othe types of base pai ings. When the RNA t ansc ipt has sepa ate f om the
r r
template, RNA synthesis can no longe contnue (see Figu e 13.13).
d d r
Rho-in epen ent te minaton
d d r r r Rho- epen ent te minato s have two featu es:
(1) DNA sequences that p o uce a pause in t ansc ipton an r d r r d
d r r
(2 ) a D NA s e q u e n c e th a t e n c o e s a s t e tc h of R N A u p s t e a m of th e
r r d d d r r r r r d r
te minato that is evoi of any secon a y st uctu es. This unst uctu e RNA se ves
d r r r d d rd
as a bin ing site fo the ho p otein, which bin s the RNA an moves towa its 3′
d r r r
en , following the RNA polyme ase (Figu e 13.14). When RNA polyme ase
r r r r r r
encounte s the te minato , it pauses, allowing ho to catch up. The ho p otein has
d r d r r
helicase actvity, which it uses to unwin the RNA–DNA hyb i in the t ansc ipton
r r r d
bubble, b inging t ansc ipton to an en .
d d r r
Rho- epen ent te minato s
d r r r r r
3 0 . T h e follow in g ia g a m e p e s e n ts a t a n s c ip ton
unit in a hypothetcal DNA molecule.
5′...TTGACA...TATAAT...3′
3 ...A AC T G T...ATAT TA...5′
Beginning with this initial sequence:
5'-TTACG-3'
The complement created by base pairing is:
3'-AATGC-5'
The reverse complement is:
5'-CGTAA-3'
And, the inverted repeat sequence is:
5'---TTACGnnnnnnCGTAA---3'
"nnnnnn" represents any number of intervening nucleotides.
Inverted repeat
RNA Splicing
r r d r
The othe majo type of mo ifcaton of euka yotc p e-mRNA r is the removal of int ons by RNA splicing. r
d r
This mo ifcaton takes place in the nucleus, befo e the RNA moves to the cytoplasm.
r r r
Splicing of p e-mRNA equi es consensus sequences.
r r r
Splicing equi es the p esence of th ee sequencesr in
r d r r rr d
the int on. One en of the int on is efe e to as the 5 splice site′ , an the othe en is thed r d 3 splice site′ ;
r
these splice sites possess sho t consensus
M r r
sequences. ost int ons in p e-mRNAs begin with GU an en withd d AG, in icatng that these d
r r d d
sequences play a c ucial ole in splicing. In ee ,
d r
changing a single nucleot e at eithe of these sites r
p events splicing.
rd r r
The thi sequence impo tant fo splicing is at the r
b anch point, which is an a enine nucleot e that d d
r d r
lies f om 18 to 40 nucleot es upst eam of the 3′
splice site
rr d r d
The sequence su oun ing the b anch point oes not
r d r
have a st ong consensus. The eleton o mutaton of
d d r r
the a enine nucleot e at the b anch point p events splicing.
Transcriptional enhancers have traditionally been described as regulatory regions of DNA that elevate basal levels of transcription by
increasing activity of the proximal promoter.
•enhancer: a short region of DNA that can increase transcription of genes
•repressor: any protein that binds to DNA and thus regulates the expression of genes by
decreasing the rate of transcription
•activator: any chemical or agent which regulates one or more genes by increasing the rate of
transcription
Each enhancer is made up of short DNA sequences called distal control elements.
Activators bound to the distal control elements interact with mediator proteins and
transcription factors.
Rifampicin- binds with Beta subunit of prokaryotic RNA polymerase. It is an inhibitor of prokaryotic transcription initiation. It binds only to bacterial RNA polymerase but not to eukaryotic RNA polymerases. Therefore, Rifampicin is a powerful drug for treatment of bacterial infections. Used for the treatment of tuberculosis and leprosy
Mitomycin- Intercalates with DNA strands, blocks transcription, used as anticancer drug.
Alpha amanitin is a molecule made from the “death cap” mushroom and is a known potent inhibitor RNA polymerase. The mechanism of action is that alpha amanitin inhibits RNA polymerase –II at both the initiation and elongation states of transcription.
