transcription lecture- maniz

8/14/2019 Transcription Lecture- Maniz

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Transcription


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Flow of Genetic Information

DNAReplication

RNATranscription

ProteinTranslation


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Transcription: productionof mRNA copy of theDNA gene.

Transcription

Eukaryote model


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Not all RNA is translated into protein:

lSome RNA is structural - e.g. ribosomal RNA (rRNA)

lSome RNA is functional - e.g. transfer RNA (tRNA)

lSome RNA is chromosomal (some viruses)

RNA

Transcription


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From which DNA strand is RNA synthesized?

Transcription usually takes place on only ONEof the DNA strands

Transcription


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5'-GTCACCCATGGAGG-3' Nontemplate strand

3'-CAGTGGGTACCTCC-5' Template strand

5'-GUCACCCAUGGAGG-3' mRNA

RNA growth always in the 5' 3' direction

5'

3'

3'

5'5' 5' 5'

5'3'

3' 3' 3'

mRNA

mRNA mRNA mRNA

DNA

DNA

Transcription


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The classification of RNA molecules

mRNA Bring information from the DNAmessenger RNA

Transcript of protein-coding geneshenceare translated into proteinNot stable


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2. rRNA Ribosomal RNA80% of total RNAComponent of ribosome

StableInvolved in protein synthesis

3. tRNA transfer RNAHas specific secondary and tertiery struc.Binds amino acid to mRNALong lifespanInvolved in protein synthesis

There is a proofreading mechanism

4. snRNA small nuclear RNAInvolved in processing RNA


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TranscriptionlThe process by which RNA moleculesare synthesized on a DNA template iscalled transcriptionlTranscription results in the synthesis

of a single stranded RNA moleculecomplementary to the DNA templatelThe ribonucleotide sequence writtenin RNA is the genetic code which is thencapable of directing the process oftranslation, which produces polypeptidechains


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Similarities and differences fromreplication

5-3 direction

Many proteinsinvolved Initiation,

elongation andtermination

Transcriptionbubble

Starts and stops atspecific sites

RNAP not DNAP

Proofreading Posttranscriptionalmodification

1 strand copies not 2

Not all transcribed

SIMILARITIESSIMILARITIES DIFFERENCESDIFFERENCES


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Prokaryotic transcription as in thebacteria E. coliRequirements for Transcription

1. Single-stranded (ss) DNAtemplate

Non-coding DNA strand acts as template

2. All 4 RNA triphosphate nucleotides

ATP, GTP,UTP,CTP (NTP)


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3. DNA dependent RNA Polymerase (a holoenzymeconsisting of subunits)

RNA polymerase/DNA dependent RNA polymerase

One in procaryoteThree in eucaryote (RNAPI-III)


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RNA Synthesis/ Transcription

Ingredients necessary for transcription

4. Bivalent ions Mg2+

5. Activators Help in binding to DNAIncrease the rate of transcription

Determines when a gene is on/offHas sequence to bind RNAP & When you change this consensus seq- you alter rate oftranscriptionWeak promoters have additional binding domains

6. Promoter


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Protein Function Seq at 35Seq at10

Sigma 70 housekeeping TTGACATATAATSigma 32 heat shockTCTCNCCCTTGAA CCCCATNTASigma 28 flagella synthesis CTAAA

CCGATAT

Consensus sequences found in promoter

7. Transcription factor


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The consensus sequence share homology indifferent genes of same organism or in one

gene or more genes of related organism

Cis acting element

Degree of RNAP binding to differentpromoters varies and due to sequencevariation in the promoter which lead tovariable in gene expression


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RNA POLYMERASE

Transcribes all proc. DNA

In euc.:

-RNA pol II* transcribes mRNA & snRNA

-RNA pol I * transcribes16S & 23S RNA (rRNA)

-RNA pol III * tRNA & 5S rRNA

-RNA pol IV * mitochondrial RNA

Also has subunit , , like the proc. RNAPGenerally all RNAP are zinc metalloenzymesNo proofreading: mistakes every 104-105 bases


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4 polypeptide subunits 2 , 1 ,1 & 1 (~500kDa)- is the holoenzyme

Subunit & form the core enzyme-provide the catalytic basis and active site fortranscription

Subunit 70- needed for transcription i.e in initiationof transcription

RNA polymerase procaryote


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The function of sigma factor the sigma subunit of RNA polymerase is an initiation factor

there are several different sigma factors in E. colithat arespecific for different sets of genes

sigma factor functions to ensure that RNA polymerase binds

stably to DNA only at promoters

sigma destablizes nonspecific binding to non-promoter DNA

sigma stabilizes specific binding to promoter DNA

this accelerates the search for promoter DNA


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What is the function of sigma factor?

s factor is critical in promoter recognition, bydecreasing the affinity of the core enzyme fornon-specific DNA sites and increasing theaffinity for the corresponding promoter

s factor is released from the RNA pol afterinitiation (RNA chain is 8-9 nt)

Is involved in opening and closing of the DNA

double helix It helps to regulate the expression of a geneset/family in different conditions


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RNA polymerase holoenzyme (+ s factor)

closed promoter complex (moderately stable) the sigma subunit binds to the 35/-10 region

once initiation takes place, RNA polymerase doesnot need very high affinity for the promoter

sigma factor dissociates from the core polymeraseafter a few elongation reactions

elongation takes place withthe core RNA polymerase

open promoter complex (highly stable) the holoenzyme has very high affinity for promoter regions because of sigma factor

s sigma can re-bind

other core enzymes

The sigma cycle

s

s


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RNA polymerase function

catalyzes the addition of 5-ribonucleoside triP

at the 3 end of growing polyribonucleotide

Select the DNA template [3-5]

Separates the two DNA strands

Recoils the DNA after transcription


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Procaryote Promoter

The start site for transcription

The 5-TATAAT-3 is conserved inE.coliand phage Pribnow Box

Orientates RNAP to move from left to right

-35 site

Is to the left of Pribnow Box

Has a 6 base conserved sequence 5-TTGACA-3

The RNAP binds to this site and initiates

transcription

-10 site


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Prokaryote Promoter Sequences

P m t


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Promoter

lconserved sequences required for specificbinding of RNA pol and transcription initiation

lRNA polymerase seeks out the consensussequences for proper orientation for binding toinitiate transcription.

lNote promoter sites have regions of similarsequences at the -35 region and -10 region.

lMinus numbers represent bases upstream of

mRNA start point, +1 is the first base in theRNA transcript.lThe -35 and -10 boxes contain consensus

sequences


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Promoter structure in prokaryotes

5 PuPuPuPuPuPuPuPu AUG

Promoter

+1 +20-7-12-31-36

5 mRNA

mRNA

TTGACAAACTGT

-35 region

TATAATATATTA

-10 region

84 79 53 45%82

TTG64

ACA79

T44

T96%

T95

A59

A51

Aconsensus sequences

-30 -10

transcription start site

Pribnow box

+1[ ]


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CAAT Box 5-GGCAATCT-3 is on the left(upstream) to the TATA Box

Mutation of this site effects the rate oftranscription

25 site

consensus seq. 5TATAAAA3

TATA BOX

Also known as the Hognes Box

Mutation doesnt effect transcription; effectsthe start site of transcription

Eucaryotic Promoter

GC Box 5-GGGCGG-3 ; functions in bindingthe RNAPII to the transcription site


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+1Transcription start site

TATA(-25)

CAAT(-75)

Eucaryotic Promoter

No sigma factor 70There are about 6 subunits (inclusive 2, 1 ,1 )General transcription factors 6 to 8 (e.g. TFIIA-J)

EnhancerTissue specific expression at the right timePresent upstream or down stream

Binds with regulatory factors


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Transcription: organization of a geneTranscription: organization of a gene

NegativeNegative

numbersnumbersPositivePositive

numbersnumbers

+1 site+1 site

Initiation of RNA chains:Initiation of RNA chains: binding of RNAP holoenzyme to promoter region

1) RNAP holoenzyme binds looselyloosely to -35-35 region (dsDNA), then

tightlytightly to the -10-10 region of dsDNA (closed promoter)


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The transcription cycle is involves a series of

events between binding of RNAP to targetgene and dissociation of RNAP and thecompleted RNA transcript from the DNA

The transcription cycle can be divided into 3

phasesc. Initiation

d. Elongation

e. termination


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Trancriptin initiation can be divided into3 stepsIn the first step the RNAP bind to a region of DNAcall promoter. In bacteria this step involves the

initiation factor call sigma which recognized variousseq within the promoter

The RNAP with sigma attached bind to the promoterin a defined orientation so the same stand always

transcribed from a given promoterThe RNAP and the promoter form a closed complex


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In the second step, closed complexundergo a transition to the open complexconformation.

The pincer in front of the RNAP clamp

down tightly downstream of the DNA.

Sigma also changed conformation and theDNA strand is seperated forming a bubble

of single stranded DNA


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lIn Step 3, once the open promotercomplex is formed, RNA Polymerase

catalyzes the insertion of the first 5ribonucleotide which is complementary tothe 1st nucleotide at the start site of theDNA template.

lNo primer is required!lSubsequent complements are inserted and

linked together by phosphodiester bonds

lAfter several ribonucleotides have beenadded, the sigma subunit dissociates andelongation proceeds


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E. coliRNA polymerase + subunit


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RNAP synthesizes several short RNAsbefore entering the elongation phase abortive initiation

Shot RNA molecules of less than tennucleotides in length

This is probably because the region of sigmapartially block the RNA exit channel.

Once this region has been ejected andpolymerase able to make an RNA longer thanten bp, a stable ternary complex is formed,consist of enzyme, DNA template andgrowing RNA chain.

This is the beginning of elongation phase

2) Localized unwinding of the two strands of DNA provides


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2) Localized unwinding of the two strands of DNA providestemplate strand for RNAP and exposes initiation site (+1)

3) unwindsunwinds dsDNA ( 17 bp) around-10 region

5) RNAP chooses correct

strand to read (templatetemplate)

6) initiates 8-9 bp then factor is releasedfactor is released Core has reduced affinity for promoterCore has reduced affinity for promoter

4) phosphodiester bondforms between the NTPs ofRNA chain


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Transcription bubble 17 bpCore completes elongation RNAP unwinds dsDNA

mRNA displaced from back RNA/DNA h brid exists

When cores loses sigmafactor it moves away from promoter


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Elongationq Core RNA polymerase adds nucleotides to form

complementary mRNA strand (transcript)q Ribonucleotides enter the active site and are

added to the growing RNA chain under theguidance of the template DNA strand

q

Only eight to nine nucleotides of the growingchain remain base-pairedq The remainder of the RNA chain is peeled off

and directed out of the enzymeq

In E. coli, 50 nucleotides/second at 37 degreesC


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During the elongation RNAP unwind the DNAin front of the enzyme , synthesized RNA,proofread RNA , dissociate RNA from theDNA and re-anneal of DNA behind theenzyme.

In contrast with DNAP, RNAP is able to dothis functions without the assistance with

other proteins


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TERMINATION

Sequences called terminator trigger the

elongating polymerase to dissociate from theDNA and release the RNA chain it has madeTwo types of termination rho- independentand rho-dependent

Rho-independent terminator also calledintrinsic terminator.

Consist of two sequence elements: a shortinverted repeat ~ 20 nucleotides followed bya stretch of about eight A:T base pair


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The RNA that result from the invertedrepeat is able to form a stem-loop

structure by base-pairing with itselfIs called a hairpin

The hairpin is believed to cause termination

by disrupting the elongation complex

The A:U base pairs are the weakest of allbase pairs, they are more easily disrupted

by the effect of stem loop on thetranscribing polymerase and allowing theRNA to dissociate from DNA

The steps in transcription:The steps in transcription: TerminationTermination


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The steps in transcription:The steps in transcription: TerminationTermination

TerminatorsTerminators exist either:IntrinsicIntrinsic to RNA strand - intrastrand base pairing (rho-independent)

ExtrinsicExtrinsic- requires an accessory protein - rhorho to stop (rho-dependent)

rho-independent terminationrho-independent termination

Terminators complementary region (G:C-rich) which forms hairpin loop

in the ssRNA RNAP pauses on UUU and falls off. Termination

rho

mRNA

G:C rich area

complementarity causes hairpin

CC

C

C

GG

G

G

UUUU

RNAP pauses on UUU region

RNAP Falls off5

3

Bound together by H bond


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Termination

Direct termination: The RNA hairpin loop of GC(inverted repeats sequences and section of Uresidues appear to serve as signal for RNApolymerase release and termination oftranscription.


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-independent Termination Inverted repeat downstream from stop

codon in DNA sequence will form hairpinin mRNA transcript

lmRNA folds around center . When RNApolymerase assembles hairpin, it pauses and

falls off

.


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.

-dependent Termination

Less well-characterised

-dependent terminators have invertedrepeat in DNA sequence, but do not haverepeated As

- protein required for termination rhoprotein binds to specific sequences referred

to as rut. rhopulls RNA polymerase off RNA


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Possible model for -dependent termination


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Eukaryotic Transcription

lTranscription in Eukaryotes is much more complex than

the bacterial system.

lThree types of RNA Polymerases exist in Eukaryotes,

each responsible for transcribing certain types of genes.lRNA Pol I, II and III (or A, B and C)

lCis acting elements involved: CAAT (-70--80 from

start)

l and TATA (-25 from start)

lenhancers and transcription factors also involved

Eucaryotic Promoter


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+1Transcription start site

TATA(-25)

CAAT(-75)

Eucaryotic Promoter

Enhancer

Tissue specific expression at the right timePresent upstream or down streamBinds with regulatory factors


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ltranscription factors bind to the DNA to assist in

initiation: TFIID, TFIIB, TFIIA, and seven others

lIn eukaryotes RNA Polymerase ll is the mRNAproducer

lhnrna: 5 methylguanosine cap added, prior to transport

out of nucleus.lpoly a tail ( several to 250) added after cap; degrades

rapidly if tail missing

lInterviening sequencesq Introns = non coding regions

q ex. collagen has 50 introns

q Histones and interferon have no introns


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Transcription initiation in eucaryote

1. Binding to the promoters

At TATA Box No sigma factor but has transcription

factors [TFIIA-J]

2. Start of transcription

TFIIA-J will dissociate when transcription is initiated

RNA polymerase open DNA helix

RNA polymerase moves across template

Transcript has similar sequence to non template strandwhere urasil replaces tiamine

RNAP will rewind the DNA that already beentranscribed

Formation of pre initiation complex


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Formation of pre-initiation complex

Complete set of general transcription factors andpolymerase which bound together at the promoter and

ready for initiation, is called the pre-initiation complex

The formation of this complex begin at TATA element

TATA element is recognized by TFIID. TBP is a

component of TFIID which binds to TATA DNA sequence.Once binding DNA, TBP extensively distorts the TATAsequence.

The resulting TBP-DNA complex provides a platform torecruit other general transcription factors andpolymerase

l ll


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Formation of the pre-initiation complex then followed bypromoter melting, ATP is required and mediated byTFIIF which has helicase like activity that can unwind

the promoter DNAIn eucaryote promoter escape involves phosphorylationof the polymerase

The large subunit of Pol II has a C-terminal domain (CTD)CTD contains a series of repeats of the heptapeptidesequence: Tyr-Ser-Pro-Thr-Ser-Pro-Ser.

(Yeast 27 repeats in the yeast polII CTD, human 52)

Addition of phosphates group helps polymerase shedmost of the general transcription factors used forinitiation, and which the enzyme leaves behind as itescape the promoter

St i bl f t i ti i iti ti l f


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Stepwise assembly of a transcription-initiation complex from

isolated RNA polymerase II (Pol II) and general transcription

factors

Once transcribed eucaryotic RNA has to be processed


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Once transcribed, eucaryotic RNA has to be processed

These processing include: capping of the 5 end of theRNA are, splicing and polyadenylation of the 3 end

The first RNA processing event is capping

Critical for efficient translation of mRNACritical for efficient translation of mRNAand transport out of nucleus.and transport out of nucleus.

Involves addition of a methylated guanine base to the 5end of the RNA by unusual 5-5 linkage

8. Removal of phosphate group from 5 end of the

transcript9. Addition of GTP

10. Modified by addition of methyl group

The Ends of Eukaryotic mRNAs:The Ends of Eukaryotic mRNAs: 5-5-CappingCapping


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yy pp gpp g

Features:Features:

Triphosphate bridgeTriphosphate bridge 5 to 5 linkage of guanine (reverse orientation)5 to 5 linkage of guanine (reverse orientation)Guanine is methylated 7 positionGuanine is methylated 7 positionFirst 2 NTs of RNA can be methylatedFirst 2 NTs of RNA can be methylated NO cap coded for in DNANO cap coded for in DNA Essential for ribosome to bind to 5-endEssential for ribosome to bind to 5-end

PolyadenylationPolyadenylation


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The final processing event and intimately linked withThe final processing event and intimately linked with

termination of the transcriptiontermination of the transcription Once polymerase has reached the end of a gene itOnce polymerase has reached the end of a gene itencounters specific sequences after being transcribedencounters specific sequences after being transcribedinto RNA, trigger the transfer of the polyadenylationinto RNA, trigger the transfer of the polyadenylation

enzyme to that RNAenzyme to that RNA

Leading to three events: cleavage of the message,Leading to three events: cleavage of the message,addition of many adenine residues to its 3 end andaddition of many adenine residues to its 3 end andtermination of transcription by polymerasetermination of transcription by polymerase

Thought to stabilize mRNA from degradationThought to stabilize mRNA from degradation Aids in efficiency of translationAids in efficiency of translation

PolyadenylationPolyadenylation


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Uses ATPUses ATP

Embedded in transcript is aEmbedded in transcript is a poly A sitepoly A site

Cuts 11-30 NT downstream ofCuts 11-30 NT downstream of

poly A sitepoly A site

RNA splicing


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RNA splicing Group 1

Introns of primary transcript in rRNA

Intron has autolytic Ribozyme Will self splice with guanosine cofactor

Group 2

mRNA and tRNA, mitochondrial and chloroplast RNA

also self splicing but no cofactor necessary Spliceosome formation

In EukaryotesIn Eukaryotes ::


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In EukaryotesIn Eukaryotes ::RNA splicingRNA splicing removal of intron sequences removal of intron sequences

ExonsExons amino acid coding regions amino acid coding regions

Expressed sequencesExpressed sequences found in both a genes found in both a genesDNA and in the mature mRNADNA and in the mature mRNA

IntronsIntrons non-amino acid coding regions non-amino acid coding regions

Intervening sequencesIntervening sequences found in a genes DNA but found in a genes DNA butnot in the mature mRNAnot in the mature mRNA (removed from the primary(removed from the primary

transcript)transcript)

R-loops which correspond to areas missing in mature RNA

Mature RNAMature RNA

Pre-RNA or DNAPre-RNA or DNA

SplicingSplicing


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SplicingSplicing

ExonsExons 50-2,000 Bp50-2,000 BpIntronsIntrons 50-100,000 Bp50-100,000 BpIntrons very common inIntrons very common in

eukaryote geneseukaryote genes

l

Gene is 2.5 million base pairs in length

How are Introns Removed?How are Introns Removed?


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How are Introns Removed?How are Introns Removed?

RNA primary transcript has all introns and exons

RNA splicingRNA splicing involves:

removal of introns stitching together of exons to form contiguous RNA very precise mechanism to protect integrity of

reading frame!

Must have mechanism in place to distinguish between

intron and exon junctionintron and exon junction

Must have enzymes to spliceenzymes to splice out the introns

Splicing of introns is usually carried out by a complex of enzymes

known as a spliceosome

Short sequences dictate the sites of splicingShort sequences dictate the sites of splicing


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Short sequences dictate the sites of splicingShort sequences dictate the sites of splicing

Process is usually carried out by a complex of proteinsProcess is usually carried out by a complex of proteins known as theknown as the spliceosomespliceosome made ofmade ofsnRNPssnRNPs ((small nuclear ribonuclear proteinssmall nuclear ribonuclear proteins))

Introns begin with 5-GU and end with 3-AGIntrons begin with 5-GU and end with 3-AG Also intron includes a branch-point sequence upstream of 3 splice siteAlso intron includes a branch-point sequence upstream of 3 splice site Key base is the Adenine of the branch siteKey base is the Adenine of the branch site


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Splice Donor siteSplice Donor site

Splice Acceptor siteSplice Acceptor site

Regulated Process:

snRNPssnRNPs cut at junction of

exon 1/ intron

loop intron and join

to branch-point AA

cut at junction of

intron/exon 2

Exons joined lariat degraded

I P k tIn Prokaryotes :


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In Prokaryotes :In Prokaryotes :

5 end of transcript has a triphosphate,5 end of transcript has a triphosphate,rather than a methylated caprather than a methylated cap

No tail at 3 endNo tail at 3 end

No intronsNo introns

Prokaryotic vs Eukaryotic Transcription


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Prokaryotic vs. Eukaryotic Transcriptionl Prokaryote

l All promoters upstream of functional gene

l Main promoter consensus sequences TATAAT (-10) and TTGACA (-35)l One RNA polymerase with subunit makes mRNA, tRNA, rRNA

l No enhancers

l mRNA is primary transcript ready to go short lifetime (just a fewminutes)

l Eukaryote

l Promoter positions differ for each polymerase- not all upstream

l Main consensus sequence TATA box (-25) and CAAT box (-60 to -120)Plants have AGGA instead of CAAT

l RNA POL I rRNA

l RNA POL II mRNA

l RNA POL III ss rRNA, tRNA

l DNA enhancer regions work with some promoters to increase transcription

l Initial product of transcription is not usable mRNA. Primary transcript mustbe processed to form mRNA. Longer lifetime (hours/days)

Review Questions


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Review Questions

lWhat are the enzymes involved in replication?lWhat are the characteristics of replication?

lDescribe the entire process of replication

noting correctly the sites, proteins andenzymes involved

lWhat is the difference if any between theprocaryotic and eucaryotic replication

process?lWhat is the function of methylation in the

regulation of replication?

transcription lecture- maniz

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