Transcription Lecture- Maniz

Transcription

Flow of Genetic Information

RNA Transcription

DNA Replication

Protein Translation

Transcription Transcription: production of mRNA copy of the DNA gene.

Eukaryote model

Transcription RNA Not all RNA is translated into protein: ❧ Some RNA is structural - e.g. ribosomal RNA (rRNA) ❧ Some RNA is functional - e.g. transfer RNA (tRNA) ❧ Some RNA is chromosomal (some viruses)

Transcription

From which DNA strand is RNA synthesized? • Transcription usually takes place on only ONE of the DNA strands

Transcription RNA growth always in the 5' → 3' direction

5'-GTCACCCATGGAGG-3' Nontemplate strand 3'-CAGTGGGTACCTCC-5' Template strand 5'-GUCACCCAUGGAGG-3' mRNA 5' 3'

3' mRNA 5' 5' mRNA 3'

5' mRNA 3' 5' mRNA 3'

3' DNA 5' DNA

The classification of RNA molecules

• mRNA

•Bring information from the DNA •messenger RNA •Transcript of protein-coding genes hence are translated into protein •Not stable

2. rRNA •Ribosomal RNA •80% of total RNA •Component of ribosome •Stable •Involved in protein synthesis 3. tRNA

4. snRNA

•transfer RNA •Has specific secondary and tertiery struc. •Binds amino acid to mRNA •Long lifespan •Involved in protein synthesis •There is a proofreading mechanism •small nuclear RNA •Involved in processing RNA

Transcription ❧The process by which RNA molecules are synthesized on a DNA template is called transcription ❧Transcription results in the synthesis of a single stranded RNA molecule complementary to the DNA template ❧The ribonucleotide sequence written in RNA is the genetic code which is then capable of directing the process of translation, which produces polypeptide chains

Similarities and differences from replication SIMILARITIES

• 5’-3’ direction • Many proteins involved • Initiation, elongation and termination • Transcription bubble • Starts and stops at specific sites

DIFFERENCES

• RNAP not DNAP • Proofreading • Posttranscriptional modification • 1 strand copies not 2 • Not all transcribed

Prokaryotic transcription as in the bacteria E. coli Requirements for Transcription

1. Single-stranded (ss) DNA template • Non-coding DNA strand acts as template

2. All 4 RNA triphosphate nucleotides •ATP, GTP,UTP,CTP (NTP)

3. DNA dependent RNA Polymerase (a holoenzyme consisting of subunits)

RNA polymerase/DNA dependent RNA polymerase One in procaryote Three in eucaryote (RNAPI-III)

RNA Synthesis/ Transcription Ingredients necessary for transcription 4. Bivalent ions

5. Activators

•Mg2+ •Help in binding to DNA •Increase the rate of transcription

6. Promoter Determines when a gene is on/off Has sequence to bind RNAP & σ When you change this consensus seq- you alter rate of transcription Weak promoters have additional binding domains

7. Transcription factor Consensus sequences found in promoter

Protein Function Seq at–10

Seq at –35

Sigma 70 housekeeping TTGACA TATAAT Sigma 32 heat shock TCTCNCCCTTGAA CCCCATNTA Sigma 28 flagella synthesis CTAAA CCGATAT

The consensus sequence share homology in different genes of same organism or in one gene or more genes of related organism Cis acting element Degree of RNAP binding to different promoters varies and due to sequence variation in the promoter which lead to variable in gene expression

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 α, β, β’ like the proc. RNAP •Generally all RNAP are zinc metalloenzymes •No proofreading: mistakes every 104-105 bases

RNA polymerase procaryote 4 polypeptide subunits 2α, 1 β,1 β’ & 1 σ (~500kDa) - is the holoenzyme Subunit α & β form the core enzyme β-provide the catalytic basis and active site for transcription Subunit σ70- needed for transcription i.e in initiation of transcription

The function of sigma factor • the sigma subunit of RNA polymerase is an “initiation factor” • there are several different sigma factors in E. coli that are specific 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

What is the function of sigma factor?

s factor is critical in promoter recognition, by decreasing the affinity of the core enzyme for non-specific DNA sites and increasing the affinity for the corresponding promoter s factor is released from the RNA pol after initiation (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 gene set/family in different conditions

s

• closed promoter complex (moderately stable) • the sigma subunit binds to the –35/-10 region RNA polymerase holoenzyme (+

s

s

factor)

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

• once initiation takes place, RNA polymerase does not need very high affinity for the promoter • sigma factor dissociates from the core polymerase after a few elongation reactions

s

• sigma can re-bind other core enzymes

• elongation takes place with the core RNA polymerase

The sigma cycle

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

Procaryote Promoter

-10 site •The start site for transcription •The 5’-TATAAT-3’ is conserved in E.coli and 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

Prokaryote Promoter Sequences

Promoter ❧ conserved sequences required for specific binding of RNA pol and transcription initiation ❧ RNA polymerase seeks out the consensus sequences for proper orientation for binding to initiate transcription. ❧ Note promoter sites have regions of similar sequences at the -35 region and -10 region. ❧ Minus numbers represent bases upstream of mRNA start point, +1 is the first base in the RNA transcript. ❧ The -35 and -10 boxes contain consensus sequences

Promoter structure in prokaryotes

[

-30

-10

Promoter

5’

+1

mRNA

PuPuPuPuPuPuPuPu

AUG

]

transcription start site

-35 region TTGACA AACTGT -36 -31

T T G AC A

82 84 79 64 53 45%

-10 region TATAAT ATATTA -12 -7 Pribnow box

T AT A AT

5’

79 95 44 59 51 96%

consensus sequences

+1

mRNA

+20

Eucaryotic Promoter •–25 site  consensus seq. 5’TATAAAA3’  TATA BOX •Also known as the Hognes Box •Mutation doesn’t effect transcription; effects the start site of transcription •CAAT Box  5’-GGCAATCT-3’ is on the left (upstream) to the TATA Box •Mutation of this site effects the rate of transcription •GC Box  5’-GGGCGG-3’ ; functions in binding the RNAPII to the transcription site

Eucaryotic Promoter TATA (-25)

CAAT (-75)

+1 Transcription start site

•No sigma factor 70 •There are about 6 subunits (inclusive 2α, 1 β,1 β’ ) •General transcription factors– 6 to 8 (e.g. TFIIA-J) Enhancer •Tissue specific expression at the right time •Present upstream or down stream •Binds with regulatory factors

Transcription: organization of a gene

+1 site Negative numbers

Positive numbers

Initiation of RNA chains:

• binding of RNAP holoenzyme to promoter region

1) RNAP holoenzyme binds loosely to -35 region (dsDNA), then tightly to the -10 region of dsDNA (closed promoter)

The transcription cycle is involves a series of events between binding of RNAP to target gene and dissociation of RNAP and the completed RNA transcript from the DNA The transcription cycle can be divided into 3 phases c. Initiation d. Elongation e. termination

Trancriptin initiation can be divided into 3 steps In the first step the RNAP bind to a region of DNA call promoter. In bacteria this step involves the initiation factor call sigma which recognized various seq within the promoter The RNAP with sigma attached bind to the promoter in a defined orientation so the same stand always transcribed from a given promoter The RNAP and the promoter form a closed complex

In the second step, closed complex undergo a transition to the open complex conformation. The pincer in front of the RNAP clamp down tightly downstream of the DNA. Sigma also changed conformation and the DNA strand is seperated forming a bubble of single stranded DNA

❧ In Step 3, once the open promoter complex is formed, RNA Polymerase catalyzes the insertion of the first 5’ ribonucleotide which is complementary to the 1st nucleotide at the start site of the DNA template. ❧ No primer is required! ❧ Subsequent complements are inserted and linked together by phosphodiester bonds ❧ After several ribonucleotides have been added, the sigma subunit dissociates and elongation proceeds

E. coli RNA polymerase + σ subunit

RNAP synthesizes several short RNAs before entering the elongation phase – abortive initiation Shot RNA molecules of less than ten nucleotides in length This is probably because the region of sigma partially block the RNA exit channel. Once this region has been ejected and polymerase able to make an RNA longer than ten bp, a stable ternary complex is formed, consist of enzyme, DNA template and growing RNA chain. This is the beginning of elongation phase

2) Localized unwinding of the two strands of DNA provides template strand for RNAP and exposes initiation site (+1) 4) phosphodiester bond forms between the NTP’s of RNA chain

3) unwinds dsDNA (≈ 17 bp) around -10 region

5) RNAP chooses correct strand to read (template) template

6) initiates ≈ 8-9 bp then σ factor is released • Core has reduced affinity for promoter

When cores loses sigma factor it moves away from promoter • Transcription bubble ≈17 bp • Core completes elongation • RNAP unwinds dsDNA • mRNA displaced from back • RNA/DNA hybrid exists

Elongation

●

●

●

●

●

Core RNA polymerase adds nucleotides to form complementary mRNA strand (transcript) Ribonucleotides enter the active site and are added to the growing RNA chain under the guidance of the template DNA strand Only eight to nine nucleotides of the growing chain remain base-paired The remainder of the RNA chain is peeled off and directed out of the enzyme In E. coli, 50 nucleotides/second at 37 degrees C

During the elongation RNAP unwind the DNA in front of the enzyme , synthesized RNA, proofread RNA , dissociate RNA from the DNA and re-anneal of DNA behind the enzyme. In contrast with DNAP, RNAP is able to do this functions without the assistance with other proteins

TERMINATION

Sequences called terminator trigger the elongating polymerase to dissociate from the DNA and release the RNA chain it has made Two types of termination rho- independent and rho-dependent Rho-independent terminator also called intrinsic terminator. Consist of two sequence elements: a short inverted repeat ~ 20 nucleotides followed by a stretch of about eight A:T base pair

The RNA that result from the inverted repeat is able to form a stem-loop structure by base-pairing with itself Is called a hairpin The hairpin is believed to cause termination by disrupting the elongation complex The A:U base pairs are the weakest of all base pairs, they are more easily disrupted by the effect of stem loop on the transcribing polymerase and allowing the RNA to dissociate from DNA

The steps in transcription: Termination • rho-independent termination

rho

Terminators • complementary region (G:C-rich) which forms hairpin loop in the ssRNA – RNAP pauses on UUU and falls off. Termination

mRNA 5’

C C C C

G G G G

G:C rich area complementarity causes hairpin

UUUU

Bound together by H bond

RNAP pauses on UUU region 3’ RNAP Falls off

Terminators exist either: • Intrinsic to RNA strand - intrastrand base pairing (rho-independent) • Extrinsic - requires an accessory protein - rho to stop (rho-dependent)

Termination

• Direct termination: The RNA hairpin loop of GC (inverted repeats sequences and section of U residues appear to serve as signal for RNA polymerase release and termination of transcription.

• ρ-independent Termination • Inverted repeat downstream from stop codon in DNA sequence will form “hairpin” in mRNA transcript

❧ mRNA folds around center . When RNA polymerase assembles hairpin, it pauses and falls off

• .

• ρ-dependent Termination • Less well-characterised • ρ-dependent terminators have inverted repeat in DNA sequence, but do not have repeated A’s • ρ- protein required for termination • rho protein binds to specific sequences referred to as rut. rho pulls RNA polymerase off RNA

Possible model for ρ-dependent termination

Eukaryotic Transcription ❧ Transcription in Eukaryotes is much more complex than the bacterial system. ❧ Three types of RNA Polymerases exist in Eukaryotes, each responsible for transcribing certain types of genes. ❧ RNA Pol I, II and III (or A, B and C) ❧ Cis acting elements involved: CAAT (-70--80 from start) ❧ and TATA (-25 from start) ❧ enhancers and transcription factors also involved

Eucaryotic Promoter TATA (-25)

CAAT (-75)

+1 Transcription start site

Enhancer •Tissue specific expression at the right time •Present upstream or down stream •Binds with regulatory factors

❧ transcription factors bind to the DNA to assist in initiation: TFIID, TFIIB, TFIIA, and seven others ❧ In eukaryotes RNA Polymerase ll is the mRNA producer ❧ hnrna: 5’ methylguanosine cap added, prior to transport out of nucleus. ❧ poly a tail ( several to 250) added after cap; degrades rapidly if tail missing ❧ Interviening sequences ● ● ●

Introns = non coding regions ex. collagen has 50 introns Histones and interferon have no introns

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 strand where urasil replaces tiamine •RNAP will rewind the DNA that already been transcribed

Formation of pre-initiation complex Complete set of general transcription factors and polymerase 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 TATA sequence. The resulting TBP-DNA complex provides a platform to recruit other general transcription factors and polymerase

Formation of the pre-initiation complex then followed by promoter melting, ATP is required and mediated by TFIIF which has helicase like activity that can unwind the promoter DNA In eucaryote promoter escape involves phosphorylation of the polymerase The large subunit of Pol II has a C-terminal domain (CTD) CTD contains a series of repeats of the heptapeptide sequence: Tyr-Ser-Pro-Thr-Ser-Pro-Ser. (Yeast 27 repeats in the yeast polII CTD, human 52) Addition of phosphates group helps polymerase shed most of the general transcription factors used for initiation, and which the enzyme leaves behind as it escape the promoter

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 These processing include: capping of the 5’ end of the RNA are, splicing and polyadenylation of the 3’ end The first RNA processing event is capping Critical for efficient translation of mRNA and transport out of nucleus. Involves addition of a methylated guanine base to the 5’ end of the RNA by unusual 5’-5’ linkage 8. Removal of phosphate group from 5’ end of the transcript 9.

Addition of GTP

10. Modified by addition of methyl group

The Ends of Eukaryotic mRNAs: 5’- Capping

Features: • Triphosphate bridge • 5’ to 5’ linkage of guanine (reverse orientation) • Guanine is methylated 7’ position • First 2 NT’s of RNA can be methylated • NO cap coded for in DNA • Essential for ribosome to bind to 5’-end

Polyadenylation •The final processing event and intimately linked with termination of the transcription • Once polymerase has reached the end of a gene it encounters specific sequences after being transcribed into RNA, trigger the transfer of the polyadenylation enzyme to that RNA •Leading to three events: cleavage of the message, addition of many adenine residues to its 3’ end and termination of transcription by polymerase •Thought to stabilize mRNA from degradation • Aids in efficiency of translation

Embedded in transcript is a poly A site

Cuts 11-30 NT downstream of poly A site

Uses ATP

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 Eukaryotes : • RNA splicing – removal of intron sequences R-loops which correspond to areas missing in mature RNA Pre-RNA or DNA Mature RNA

Exons – amino acid coding regions “Expressed sequences” found in both a gene’s DNA and in the mature mRNA Introns – non-amino acid coding regions “Intervening sequences” found in a gene’s DNA but not in the mature mRNA (removed from the primary transcript)

Splicing

Gene is 2.5 million base pairs in length

Exons ≈ 50-2,000 Bp Introns ≈ 50-100,000 Bp Introns very common in eukaryote genes l

How are Introns Removed? • RNA primary transcript has all introns and exons RNA 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 junction Must have enzymes 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 splicing

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

• Process is usually carried out by a complex of proteins • known as the spliceosome • made of snRNPs (small nuclear ribonuclear proteins)

Splice Acceptor site Splice Donor site Regulated Process: snRNPs • cut at junction of exon 1/ intron • loop intron and join to branch-point A • cut at junction of intron/exon 2 • Exons joined • lariat degraded

In Prokaryotes : 5’ end of transcript has a triphosphate, rather than a methylated cap No tail at 3’ end No introns

Prokaryotic vs. Eukaryotic Transcription ❧ ❧ ❧ ❧ ❧ ❧

Prokaryote All promoters upstream of functional gene Main promoter consensus sequences TATAAT (-10) and TTGACA (-35) One RNA polymerase with σ subunit makes mRNA, tRNA, rRNA No enhancers mRNA is primary transcript – “ready to go” – short lifetime (just a few minutes)

❧ Eukaryote ❧ Promoter positions differ for each polymerase- not all upstream ❧ Main consensus sequence TATA box (-25) and CAAT box (-60 to -120) Plants have AGGA instead of CAAT ❧ RNA POL I – rRNA ❧ RNA POL II – mRNA ❧ RNA POL III – ss rRNA, tRNA ❧ DNA enhancer regions work with some promoters to increase transcription ❧ Initial product of transcription is not usable mRNA. Primary transcript must be processed to form mRNA. Longer lifetime (hours/days)

Review Questions ❧ What are the enzymes involved in replication? ❧ What are the characteristics of replication? ❧ Describe the entire process of replication noting correctly the sites, proteins and enzymes involved ❧ What is the difference if any between the procaryotic and eucaryotic replication process? ❧ What is the function of methylation in the regulation of replication?