5 Transcription

7/18/2019 5 Transcription

http://slidepdf.com/reader/full/5-transcription 1/74

Transcription

From DNA to RNA

108/06/15



What is transcription?• During transcription, an enzyme system converts the

genetic information in a segment of doublestrandedDNA into an RNA strand !ith a base se"uencecomplementary to one of the DNA strands#

DNA Dependent synthesis of RNATranscription resembles replication in its fundamental

chemical mechanism, its polarity (direction of synthesis), andits use of a template. And like replication, transcription has

initiation, elongation, and termination phases.Transcription differs from replication in that it does notrequire a primer and, generally, involves only limitedsegments of a DNA molecule. Additionally, ithin transcribedsegments only one DNA strand serves as a template for a

particular !NA molecule. 208/06/15



$ort ons o DNA %e"uence Are Transcr einto RNA

• RNA is a

linear polymermade of fourdi&erenttypes ofnucleotide

subunitslin'edtogether byphosphodiester bonds

A short length of

!NA. The

phosphodiester

chemical linkagebeteen

nucleotides in

!NA is the same

as that in DNA.

308/06/15



408/06/15



508/06/15



• like DNA, !NA contains the bases adenine (A), guanine ("), andcytosine (#), it contains the base uracil ($) instead of thethymine (T) in DNA. %ince $, like T, can base&pair by hydrogenbonding ith A.

• An !NA chain can therefore fold up into a particular shape, 'ust

as a polypeptide chain folds up to form the final shape of aprotein.

• !NA is largely single&stranded, but it often contains shortstretches of nucleotides that can form conventional base pairsith complementary sequences found elsehere on the samemolecule.

• These interactions, are non&conventional base&pairinteractions and allo an !NA molecule to fold into a three&dimensional structure that is determined by its sequence ofnucleotides

608/06/15



708/06/15



808/06/15



908/06/15



#oding strand and template strand

• The to complementary DNA strands have different roles

in transcription.

• The strand that serves as template for !NA synthesis is

called the template strand.

• The DNA strand complementary to the template is knon

as the nontemplate strand or coding strand

• #oding strand is identical in base sequence to the !NA

transcribed from the gene, ith $ in the !NA in place of T

in the DNA

1008/06/15



The to complementary strands of DNA are defined

by their function in transcription. The !NA transcript is synthesi*ed on the templatestrand and is identical in sequence (ith $ in place of

T) to the non&template strand, or coding strand.

1108/06/15



+rgani*ation of coding information in the

adenovirus genome

• The genetic information of the adenovirus genome is encoded by a

double&stranded DNA molecule of -, bp, both strands of hichencode proteins.

• The information for most proteins is encoded by (that is, identical to) the

top strand by convention, the strand oriented /0 to 0 from left to right.

• The bottom strand acts as template for these transcripts.

• 1oever, a fe proteins are encoded by the bottom strand, hich is

transcribed in the opposite direction (and uses the top strand as

template).

1208/06/15



RNA (s %ynthesized by RNA$olymerases

•

DNA dependent !NA polymerase requires a DNA template, allfour ribonucleoside /0&triphosphates (AT2, "T2, $T2, and

#T2) as precursors of the nucleotide units of !NA and 3g 45 .

• !NA polymerase elongates an !NA strand by adding

ribonucleotide units to the 0&hydro6yl end, building !NA in

the /07 0 direction.

• The 0 hydro6yl group acts as a nucleophile, attacking the ᾳ

phosphate of the incoming ribonucleoside triphosphate and

releasing pyrophosphate. The overall reaction is&

(N32)n 5 NT27 (N32)n58 5 22i

!NA 9engthened !NA

1308/06/15



#atalytic mechanism of !NA synthesis by !NA

polymerase

The reaction involves to 3g45 ions, coordinated to the

phosphate groups of the incoming NT2 and to three Asp

residues (Asp:-. , Asp:-4 and Asp:-: in the beta ( ᵝ ) subunit of the

E. coli !NA polymerase), hich are highly conserved in the!NA polymerases of all species.

+ne 3g45 ion facilitates attack by the 0 hydro6yl group on the

alpha phosphate of the NT2,

The other 3g

45

ion facilitates displacement of thepyrophosphate

;oth metal ions stabili*e the pentacovalent transition state

1408/06/15



1508/06/15



T!AN%#!<2T<+N <N 2!+=A!>+T?%

• E. coli !NA polymerase consists of α, β , β 0, ω, and σ subunits.

!NA 2olymerase and Transcription

1608/06/15



%ignals ?ncoded in DNA Tell !NA

2olymerase @here to %tart and %top

•

The bacterial !NA polymerase core en*yme is a multisubunitcomple6 that sythesi*es !NA using a DNA template as a guide.

%igma () factorB Transcription is initiated by the binding of

σ to promoter sequences.

•

A detachable subunit called sigma () factor associates ith the coreen*yme and assists it in reading the signals in the DNA that tell it

here to begin transcribing.

• factor and core en*yme are knon as the !NA polymerase

holoen*yme.

• After synthesis of about the first ten nucleotides of !NA, the core

polymerase dissociates from σ and travels along the template DNA as

it elongates the !NA chain

1708/06/15



2romoterB a special sequence of nucleotides indicating

the starting point for !NA synthesis

• !NA polymerase recogni*es the promoter as double&

stranded DNA.

@hen the polymerase holoen*yme slides into a region on

the DNA double heli6 called a promoter the polymerase

binds tightly to this DNA.

The polymerase holoen*yme, through its factor,

recogni*es the promoter DNA sequence by making specificcontacts ith the portions of the bases that are e6posed on

the outside of the heli6

%ignals ?ncoded in DNA Tell !NA

2olymerase @here to %tart and %top

1808/06/15



#onsensus sequence

• The promoters are characteri*ed by to he6americ DNA sequences,

the C/ sequence and the C8 sequence named for theirappro6imate location relative to the start point of transcription

(designated 58).

• Analyses and comparisons of the most common class of bacterial

promoters have revealed similarities in to short sequences centered

about positions &8 and & /.

• #ertain nucleotides that are particularly common at each position

form this consensus sequence.

• The consensus sequence at the & 8 region is (/0)TATAAT(0) the

consensus sequence at the & / region is (/0) TT"A#A (0)

• These sequences are important interaction sites for the subunit.

1908/06/15



2008/06/15



Transcription then continues until the

polymerase encounters a termination signal

2108/06/15



TerminatorB a second signal in the DNA, here the polymerase

halts and releases both the nely made !NA chain and the DNA

template

After the polymerase core en*yme has been released at a terminator, itre associates ith a free sigma factor to form a holoen*yme that can

begin the process of transcription again.

1o do the termination signals in the DNA stop the elongating

polymeraseE

• For most bacterial genes a termination signal consists of a string of AC T nucleotide pairs ith a to&fold symmetric DNA sequence. During

transcription, it folds into a hairpin structure through @atsonC

#rick base&pairing.

• As the polymerase transcribes across a terminator, the formation of

the hairpin may help to pull the !NA transcript from the activesite.

2208/06/15



• The DNAC!NA hybrid in the active site, is held together

at terminators predominantly by $CA base pairs (hich

are less stable than "C# base pairs because they form to

rather than three hydrogen bonds per base pair),

• $CA base pairs are not strong enough to hold the !NA in

place, and it dissociates causing the release of the

polymerase from the DNA

2308/06/15



Transcription by !NA polymerase in E. coli

• For synthesis of an !NA strand complementary to one of to

DNA strands in a double heli6, the DNA is transiently unound.

• About 8 bp are unound at any given time.

• !NA polymerase and the transcription bubble move from left

to right along the DNA , facilitating !NA synthesis.

• The DNA is unound ahead and reound behind as !NA is

transcribed. !ed arros sho the direction in hich the DNA

must rotate to permit this process.

•As the DNA is reound, the !NA&DNA hybrid is displaced andthe !NA strand e6truded.

• The !NA polymerase is in close contact ith the DNA ahead of

the transcription bubble, as ell as ith the separated DNA

strands and the !NA ithin and immediately behind the bubble.2408/06/15



2508/06/15



2608/06/15



The transcription cycle of bacterial !NA

polymerase

• <n step 8, the !NA polymerase holoen*yme (polymerase

core en*yme5 factor) assembles and then locates a

promoter

• The polymerase uninds the DNA at the position at hich

transcription is to begin (step 4)

• And begins transcribing (step ).

• @hen !NA polymerase has managed to synthesi*e about

8 nucleotides of !NA, it breaks its interactions ith the

promoter DNA and factor. The polymerase no shifts to

the elongation mode of !NA synthesis (step :).

• <t moves rightard along the DNA in this diagram. During

the elongation mode (step /).2708/06/15



• The polymerase leaves the DNA template and releases the

nely transcribed !NA only hen it encounters a

termination signal (steps - and ).

• Termination signals are typically encoded in DNA, and

many function by forming an !NA structure that

destabili*es the polymeraseGs hold on the !NA (step ).

2808/06/15



The transcription cycle of bacterial !NA polymerase

2908/06/15

Th i t f !NA l i t ti



The importance of !NA polymerase orientation

• A gene typically has only a single promoter, and because thepromoterGs nucleotide sequence is asymmetric the polymerasecan bind in only one orientation.

• The polymerase synthesi*es !NA in the /0&to&0 direction and

it can therefore only transcribe one strand per gene 3008/06/15



Directions of transcription along a short

portion of a bacterial chromosome

• "enome sequences reveal that the DNA strand used as the

template for !NA synthesis varies from gene to gene

depending on the location and orientation of the promoter.

3108/06/15



%teps of transcription

)

Ne*3208/06/15



Ne*3308/06/15



+

Ne*3408/06/15



-

.

Ne*3508/06/15



/

Ne*3608/06/15



0

Ne*3708/06/15



3808/06/15



Transcription <nitiation in ?ucaryotes !equires

3any 2roteins

• ;acteria contains a single type of !NA polymerase

• ?ucaryotic nuclei have threeB !NA polymerase <, !NA

polymerase <<, and !NA polymerase <<<. The three

polymerases are structurally similar to one another (and tothe bacterial en*yme) and share some common subunits,

but they transcribe different types of genes.

• !NA polymerases < and <<< transcribe the genes encoding

transfer !NA, ribosomal !NA, and various small !NAs.• !NA polymerase << transcribes most genes, including all

those that encode proteins

3908/06/15



4008/06/15

%tructural similarity beteen a bacterial !NA



%tructural similarity beteen a bacterial !NA

polymerase and a eucaryotic !NA polymerase <<

!egions of the to !NApolymerases that have similar

structures are indicated in green.

The eucaryotic polymerase is

larger than the bacterial en*yme

(84 subunits instead of /),The blue spheres represent Hn

atoms that serve as structural

components of the polymerases,

and

The red sphere represents the3g atom present at the active

site, here polymeri*ation takes

place.

4108/06/15



several important differences in the function of

bacterial and eucaryotic !NA polymerase

en*ymes8. ;acterial !NA polymerase requires only a single additional

protein ( factor) for transcription initiation to occur in

vitro, eucaryotic !NA polymerases require many additional

proteins, collectively called the general transcriptionfactors.

4. ?ucaryotic transcription initiation must deal ith the

packing of DNA into nucleosomes and higher&order forms

of chromatin structure, features absent from bacterialchromosomes.

4208/06/15



!NA 2olymerase << !equires "eneral

Transcription Factors

• "eneral transcription factorsB

1elp to position eucaryotic !NA polymerase correctly at the

promoter

Aid in pulling apart the to strands of DNA to allo

transcription to begin,

!elease !NA polymerase from the promoter into the elongation

mode once transcription has begun.

• The proteins ( transcription factors) are general because they

are needed at nearly all promoters used by !NA polymerase <<I

• The proteins consist of a set of interacting proteins, they are

designated as TFII ( Transcription Factor for polymerase <<)

and are denoted arbitrarily as TF<<;, TF<<D and so on.

4308/06/15



• <n a broad sense, the eucaryotic general

transcription factors carry out functions

equivalent to those of the factor in

bacteria

• 2ortions of TF<<F have the same three&

dimensional structure as the equivalent

portions of .

4408/06/15

<nitiation of transcription of a eucaryotic gene



<nitiation of transcription of a eucaryotic gene

by !NA polymerase <<

• To begin transcription, !NA polymerase requires several general

transcription factors.

(A) The promoter contains a DNA sequence called the TATA bo6,

hich is located 4/ nucleotides aay from the site at hich

transcription is initiated.

(;) Through its subunit T;2, TF<<D recogni*es and binds theTATA bo6,

(#) <t enables the ad'acent binding of TF<<;.

(D) The rest of the general transcription factors, as ell as the

!NA polymerase itself, assemble at the promoter. (?) TF<<1 then uses AT2 to pry apart the DNA double heli6 at

the transcription start point, locally e6posing the template strand.

TF<<1 also phosphorylates !NA polymerase <<, changing its

conformation so that the polymerase is released from the general

factors and can begin the elongation phase of transcription. 4508/06/15



<nitiation of

transcription of

a eucaryotic geneby !NA

polymerase <<

4608/06/15



4708/06/15



4808/06/15



4908/06/15



As shon, the site of phosphorylation is a long #&

terminal polypeptide tail, also called the #&

terminal domain (#TD), that e6tends from the

polymerase molecule. !NA polymerase << isphosphorylated at this tail.

transcription factors have been highly conserved

in evolutionI some of those from human cells can

be replaced in biochemical e6periments by thecorresponding factors from simple yeasts.

5008/06/15



5108/06/15



• TATA bo6 B "eneral transcription factor TF<<D binds to a

short double&helical DNA sequence primarily composed of

T and A nucleotides. This sequence is knon as the TATA

sequence, or TATA bo6. The TATA bo6 is typically located

4/ nucleotides upstream from the transcription start site.<t is not the only DNA sequence that signals the start of

transcription.For most polymerase << promoters it is most

important.

•T;2B TATA binding proteinB the subunit of TF<<D thatrecogni*es TATA bo6 is called TATA binding protein.

• The binding of TF<<D causes a large distortion in the DNA

5208/06/15

Three&dimensional structure of T;2 (TATA&binding



( g

protein) bound to DNA

5308/06/15

2olymerase << Also !equires Activator



2olymerase << Also !equires Activator,

3ediator, and #hromatin&3odifying 2roteins

• Transcription initiation in a eucaryotic cell is more comple6

• "ene regulatory proteins knon as transcriptional activators

must bind to specific sequences in DNA and help to attract

!NA polymerase << to the start point of transcription

• These proteins bind to specific short sequences in DNA.• These gene regulatory proteins help !NA polymerase, the

general transcription factors, and the mediator all to

assemble at the promoter.

•<n addition, activators attract AT2&dependent chromatinremodeling comple6es and histone acetylases.

• #hromatin is probably the &nm filament.

• And should be a form of DNA upon hich transcription is

initiated 5408/06/15



5508/06/15



5608/06/15



5708/06/15



5808/06/15



Transcription ?longation 2roduces %uper

helical Tension in DNA

• 1longating RNA polymerases, both bacterialand eucaryotic, are associated !ith a series ofelongation factors, proteins that enables RNApolymerase not to dissociate before it reaches

the end of a gene#• DNA supercoiling represents a conformation

that DNA adopts in response to superhelicaltension2

• creating various loops or coils in the heli* cancreate such tension#

• There are appro*imately )3 nucleotide pairsfor every helical turn in a DNA double heli*

5908/06/15



6008/06/15



6108/06/15



• A moving polymerase generates positive superhelical

tension in the DNA in front of it and negative helicaltension behind it.

6208/06/15

Transcription ?longation in ?ucaryotes <s Tightly



#oupled to !NA 2rocessing

• Transcription is only the first of several steps needed to produce

an m!NA.• +ther critical steps are the covalent modification of the ends of

the !NA and the removal of intron sequences that are discarded

from the middle of the !NA transcript by the process of !NA

splicing

• <n eucaryotic cells the !NA molecule resulting from transcription

contains both coding (e6on) and noncoding (intron) sequences.

;efore it can be translated into protein, the to ends of the !NA

are modified, the introns are removed by an en*ymatically

cataly*ed !NA splicing reaction, and the resulting m!NA istransported from the nucleus to the cytoplasm

• The !NA cap is added and splicing typically begins before

transcription has been completed

6308/06/15



6408/06/15



2rocessing of m!NA (pre&m!NA) in prokaryotes

• <n procaryotes the production of m!NA is muchsimpler.

• The /0 end of an m!NA molecule is produced by the

initiation of transcription, and the 0 end is produced by

the termination of transcription.• %ince procaryotic cells lack a nucleus, transcription and

translation take place in a common compartment.

• <n fact, the translation of a bacterial m!NA often begins

before its synthesis has been completed

6508/06/15



6608/06/15

A comparison of the structures of procaryotic



p p y

and eucaryotic m!NA molecules

• ;oth ends of eucaryotic m!NAs are modifiedB by capping on

the /0 end and by cleavage of the pre&m!NA transcript and

polyadenylation of the 0end.

addition of a poly&A tail J polyadenylation

• The /0end 0ends of a bacterial m!NA are the unmodified

ends of the chain synthesi*ed by the !NA polymerase.

• Another difference beteen the procaryotic and eucaryotic

m!NAsB bacterial m!NAs can contain the instructions for

several different proteins,

• @hereas eucaryotic m!NAs nearly alays contain the

information for only a single protein.

6708/06/15



6808/06/15



The structure of the cap at the /0 end of

eucaryotic m!NA molecules

• $nusual /0&to&/0 linkage of the &methyl " to the

remainder of the !NA.

•3any eucaryotic m!NAs carry an additionalmodificationB the 40&hydro6yl group on the second ribose

sugar in the m!NA is methylated

6908/06/15



7008/06/15

!NA %plicing !emoves <ntron %equences



!NA %plicing !emoves <ntron %equences

from Nely Transcribed 2re&m!NAs

• ?ucaryotic genes ere found to be broken up into small

pieces of coding sequence (e6pressed sequences or e6ons).

• ?6ons are interspersed ith much longer intervening

sequences or introns.

• Thus, the coding portion of a eucaryotic gene is often only

a small fraction of the length of the gene.

7108/06/15



7208/06/15



• The intron se"uences are removed from thene!ly synthesized RNA through the processof RNA splicing#

• 4nly after .5and 5 end processing andsplicing, precursormRNA 6or premRNA7 isconverted to mRNA

7308/06/15



Documents

5 Transcription