TRANSCRIPTION

TRANSCRIPTION

Transcription Prokaryotic transcription The RNA polymerase The origin & prokaryotic promoters The initiation, elongation,& termination. Prokaryotic termination signals Prokaryotic transcription product Eukaryotic transcription Eukaryotic RNA polymerases Eukaryotic promoters

Enhancers & transcriptional elements The processes of initiation, elongation & termination Eukaryotic termination signals RNA processing & modificationsThe spliceosomesThalassemias & globin mRNA splicingModification to mRNA, tRNA

The CentralDogma DNA codes for RNA RNA codes for protein

Transcription: synthesis of one RNAmolecule using one of the two DNAstrands as a template by the enzymeRNA Polymerase.Newly Synthesized RNA

The RNA polymerase-catalyzed synthesis ofRNA on a DNA template strand

Eukaryotic Transcription and Tanslation are separatedby space and ngsplicingmRNAnuclear exportcytosoltranslation

RNA Polymerase The enzyme responsible for the RNAsynthesis is DNA-dependent RNApolymerase.–The prokaryotic RNA polymeraseis a multiple-subunit protein of 480kD.

RNA Polymerase(NMP)n NTP (NMP)n 1 PPi1. Requires no primer for polymerization.2. Requires DNA for activity and is mostactive with a double-stranded DNA astemplate.3. 5’ 3’ synthesis.4. Require Mg2 for RNA synthesis activity.5. lacks 3’ 5’ exonuclease activity, and theerror rate of nucleotides incorporation is10-4 to 10-5.6. Usually are multisubunit enzyme.

RNA Polymerase of E. ColiThe holoenzyme of RNA-polymerase in E.coliconsists of 5 different subunits: 2 SubunitMWFunction 36.5 KDDetermines the DNA to betranscribed 150 KDCatalyzes polymerization 155.5 KDBinds & open DNA template 70 KDRecognizes the promoterfor synthesis initiationω11 KDSubunit packing

Eukaryotic Promoter Sequences Promoter Enhancers Activators

Structure of bacterial prokaryoticpromoter regionTATA-Box / Pribnow box This is a stretch of 6nucleotides ( 5'- TATAAT-3')centered about 8-10nucleotides to the left of thetranscription start site.-35 Sequence A second consensusnucleotide sequence( 5'- TTGACA-3'), is centeredabout 35 bases to the left ofthe transcription start site.Biochemistry For Medics- Lecture Notes17

Transcription process1. Promoter binding2. DNA unwinding3. RNA chain initiation4. RNA chain elongation5. RNA chain termination

Transcription of Prokaryotes Initiation phase: RNA-polymeraserecognizes the promoter and startsthe transcription. Elongation phase: the RNA strandis continuously growing. Termination phase:RNA-polymerase stops synthesisand the nascent RNA is separatedfrom the DNA template.

Initiation of Transcription atPromotersTranscription is divided into three steps forboth prokaryotes and eukaryotes.Initiation, Elongation and Termination.The process of elongation is highlyconserved between prokaryotes andeukaryotes, but initiation andtermination are somewhat different.

Initiation RNA-polymerse recognizes theTTGACA region (-35 sequence), andslides to the TATAAT region(-10 sequence), then opens the DNAduplex. The unwound region is about 17 bp.

The first nucleotide on RNA transcriptis always purine triphosphate. GTP ismore often than ATP. The pppGpN-OH structure remains onthe RNA transcript until the RNAsynthesis is completed. The three molecules form atranscription initiation complex.RNA-pol ( 2 ) - DNA - pppGpN- OH 3

No primer is needed for RNA synthesis. The subunit falls off from theRNA-polymerase once the first3 ,5 -phosphodiester bond is formed. The core enzyme moves along the DNAtemplate to enter the elongation phase.

Elongation The release of the subunit causesthe conformational change of thecore enzyme. The core enzyme slideson the DNA template toward the 3 end. Free NTPs are added sequentially tothe 3 -OH of the nascent RNA strand.(NMP)n NTPRNA strandsubstrate(NMP)n 1 PPielongatedRNA strand

RNA-polymerase, DNA segment of 40nt and the nascent RNA form acomplex called the transcriptionbubble. The 3 segment of the nascent RNAhybridizes with the DNA template, andits 5 end extends out thetranscription bubble as the synthesisis processing.

Transcription bubbleNascent RNA

RNA Polymerase

Removal ofSigma subunitNascent RNA

Hairpin5’RNA3’

Termination The RNA Polymerase stops moving onthe DNA template. The RNA transcriptfalls off from the transcription complex. The termination occurs in either -dependent or -independent manner.

Termination function of factor -dependent terminationThe factor, a hexamer, is a ATPaseand a Helicase.

-independent termination The termination signal is a stretch of30-40 nucleotides on the RNAtranscript, consisting of many GCfollowed by a series of U. The sequence specificity of thisnascent RNA transcript will formparticular stem-loop structures toterminate the transcription.

-independent terminationHairpin

Synthesis of rRNA and tRNACleavage of this transcript produces 5S,16S, and 23S rRNA molecules and a tRNAmolecule.Spacer regions are shown in yellow.

INHIBITORS OF TRANSCRIPTION

It binds to the β-subunit of the RNA Polymeraseto block the initiation of transcription.

It binds with the β-subunit of prokaryoticRNA Polymerase and thus inhibits theElongation phase of Transcription.

Actinomycin DThe tricyclic ring system (phenoxazone) ofActinomycin D intercalates between adjacentG-C base pairs, and the cyclic polypeptidearms fill the nearby narrow groove andinhibits Elongation phase of Transcription.

Cordycepin (3-deoxy Adenosine)It inhibits the Elongation phase of Transcription

EUKARYOTIC TRANSCRIPTION

Eukaryotic RNA polymerasesRNA Polymerase-I: Transcribes / Synthesizes 28s rRNA 18s rRNA 5.8s rRNARNA Polymerase-II: Transcribes m-RNA Some sn-RNARNA Polymerase-III: Transcribes t-RNA 5s rRNA Some sn-RNA

RNA POLYMERASE-II RNA polymerase II is central to eukaryoticgene expression and has been studiedextensively. RNA polymerase II is a multi subunitenzyme with 12 subunits. RNA polymerase II requires an array ofother proteins, called transcription factors(TF II) in order to form the activetranscription complex.

Eukaryotic RNA polymerases

α-Amanitin ( Fungal toxin from Amanitaphalloides) - cyclic octapeptide withunussual amino acids.Inhibitor of eukaryotic RNA polymerase(mainly of type II )

Eukaryotic TranscriptionPromoters Much more complex than those found in bacteria. These are consensus sequences located at theupstream regions of Coding strand. Mutation of this region usually significantly lowersthe rate of transcription.

1) TATA box ( Hogness Box)Very similar to the prokaryotic TATA box,except the sequence is slightly different(TATAAA) and it is located in between-25 to -30.2) CAAT boxLocated in between -70 to -80.Always contains CCAAT.3) GC boxUsually has the sequence GGGCGGand is typically found at -110.

ENHANCERS : Enhancers elements are thesequences located in a variety ofregions of a gene both upstreamand downstream of thetranscription start site and evenwithin the transcribed portions ofsome genes. Enhancers increases thetranscription rate by several folds.

UPSTREAMDOWNSTREAM

Transcription factors RNA-pol II does not bind to thepromoter sequences directly. RNA-pol II associates with sixtranscription factors. TFII A, TFII B, TFII D, TFII E, TFII Fand TFII H

Pre-initiation complex (PIC) TBP of TFII D binds TATA–Box(-10 sequence)TFII A and TFII B bind TFII DTFII F- RNA-pol complex binds TFII BTFII F and TFII E open the dsDNA (helicaseand ATPase) TFII H: completion of PICRNA pol IITF II FTF IIATBP TF IIDTATATF IIBTF II ETF II HDNA

Pre-initiation complex (PIC)RNA pol IITF II F5’3’TF IIATBPTF II DTATATF IIBTF II E3’TF II HDNA5’DNA RNA Poly-II TBP Transcription Factors (TF)

Phosphorylation of RNA-Polymerase-II TF II H is of protein kinase activity tophosphorylate CTD of RNA pol-II.(CTD is the C-terminal domain of RNA pol-II) Only the RNA Polymerase can move towardthe downstream, starting the elongationphase. Most of the Transcription Factors fall offfrom PIC during the elongation phase.

RNA Poly-IICTD

Termination When the RNA Polymerase transcribes theterminator region of the DNA, the polymerasereleases the mRNA The termination sequence is AATAAA followedby GT repeats.

ElongationTFIIF remains associated with RNA Pol-IIthroughout elongation.The activity of the RNA poly-II is greatlyenhanced by proteins called Elongation factors

INHIBITORSOFEUKARYOTIC TRANSCRIPTION

Mitomycin Mitomycin- Intercalateswith DNA strands Blocks transcription Used as anticancer drug

ADRIAMYCINInhibits the Initiation phase by preventing theinteraction of TF-IID with RNA-Poly-II andDNA complex.

CYCLOSPORIN -AImmunosuppressant Drug inhibits Transcriptionin T-Cells.

e)Inhibits the Elongation phase of Transcriptionby selectively inhibiting RNA Poly-II.

Flavopyridol (Alvocidib)Inhibits the Elongation phase of Transcriptionby selectively inhibiting RNA Poly-II.

TagetitoxinInhibits tRNA synthesis by binding toRNA Poly-III.

Post-TranscriptionalModifications

The nascent RNA, also known asPrimary transcript, needs to bemodified to become functional, mRNAs,tRNAs and rRNAs. These modification is critical toeukaryotic systems.

Modification of hnRNA Primary transcripts of mRNA are called asheteronuclear RNA (hnRNA). hnRNA are larger than matured mRNA by manyfolds. Modification includes– Capping at the 5 - end– Tailing at the 3 - end– mRNA splicing– RNA editing

Post Transcriptional modifications ofPre-mRNA (or) hnRNAExons Introns are removed from the primarytranscript in the nucleus, exons (codingsequences) are ligated to form the mRNAmolecule.

Capping at the 5 - endOHOHONH2NNONOOONH5'H2C O P O P O P O CH25'HNOONOONNH 2NOCH 3PiOOP OOm7GpppGp----OHAAAAA-OH 3'

5' Cap

The 5 - cap structure is found on hnRNAtoo. The capping process occurs innuclei. The cap structure of mRNA will berecognized by the cap-binding proteinrequired for translation. The capping occurs prior to the splicing.

Poly-A tailing at 3 - end

mRNA splicingmRNADNAThe matured mRNAs are much shorter thanthe DNA templates.

Split geneThe structural genes are composed of Coding(Exons) and Non-coding (Introns)regionsthat are alternatively separated.7 700 bpL1A234B C DA G no-coding region5E6F7G1 7 coding region

Splicing of hnRNA / pre-mRNA Introns (or) intervening sequences are the RNAsequences which do not code for the proteins. Introns usually start with 5 -GU. Introns usually end with 3 -AG. RNA splicing involves the removal intronsfrom pre-mRNA and is carried out by smallnuclear complexes Spliceosomes.

Spliceosome The spliceosome is a large Protein-RNAcomplex in which splicing of pre-mRNAsoccurs. The spliceosome is made up of specializedRNA and Protein complexes called smallnuclear RiboNucleoProteins (snRNPs, oftenpronounced “snurps”). Each snRNP contains RNAs with 100 to 200nucleotides long, known as small nuclearRNAs (snRNAs).

Five snRNAs (U1, U2, U4, U5, and U6)involved in splicing reactions are generallyfound in abundance in eukaryotic nuclei. Splice sites of Introns are recognized bysnRNPs.84

Intron14253Exon 1Exon 2

Self splicing Introns (Group –I Introns)IntronExon 1Exon 2

Self splicing Introns (Group –II Introns)

Eukaryotic m-RNA after modifications

Splice site mutations Mutation at splice sites can lead to impropersplicing and production of abberant proteins Eg: - thalassemia β-subunit of hemoglobin is not formed insufficient amount. It results from point mutation in -globingene where the G A mutation occurs. This creates a new splice acceptor sitenineteen nucleotides upstream from thenormal splice acceptor A faulty beta-globin protein is made, leadingto severe anemia.89

Location of Globin genes

Distribution of β-globin gene mutations associatedwith β-thalassemia.

World distribution of β-thalassemia

Clinical syndromes intermediaSevere; requires bloodtransfusionsSevere but does notrequire regular bloodtransfusionsβ-Thalassemia Asymptomatic with mildminoror absent anemia; red cellabnormalities seen

Modification of tRNA

Endo- and exonucleases to generateends of tRNA Endonuclease RNase P cleaves to generate the5 end. Exonuclease RNase D trims 3′ to 5′, leaving themature 3 end.95