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TRANSCRIPTION Dr. Harinikumar. K.M. University of Agricultural sciences Bengaluru Karnataka. www.powerpointpresentationon.blogspot.com. The Central Dogma. The central dogma of molecular biology was first articulated by Francis Crick in 1958 and re-stated in a Nature paper published in 1970

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TRANSCRIPTION Dr. Harinikumar. K.M. University of Agricultural sciences Bengaluru Karnataka

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Transcription dr harinikumar k m university of agricultural sciences bengaluru karnataka

TRANSCRIPTIONDr. Harinikumar. K.M.University of Agricultural sciences Bengaluru Karnataka

www.powerpointpresentationon.blogspot.com


The central dogma

The Central Dogma

The central dogma of molecular biology was first articulated by Francis Crick in 1958 and re-stated in a Nature paper published in 1970

The central dogma of molecular biology deals with the detailed residue-by-residue transfer of sequential information. It states that such information cannot be transferred back from protein to either protein or nucleic acid.

In other words, the process of producing proteins is irreversible: a protein cannot be used to create DNA.


Transcription dr harinikumar k m university of agricultural sciences bengaluru karnataka

Prokaryotes : Relatively Simple….


Transcription dr harinikumar k m university of agricultural sciences bengaluru karnataka

Eukaryotes : Not so Simple….


Introduction

Introduction

  • The majority of genes are expressed as the proteins they encode. The process occurs in two steps:

    • Transcription = DNA → RNA

    • Translation = RNA → protein


Terminology related to transcription

Terminology Related To Transcription

  • Coding strand (or sense strand or non-template strand)-The DNA strand that has the same sequence as the mRNA and is related by the genetic code to the protein sequence that it represents.

  • RNA polymerase – An enzyme that synthesizes RNA using a DNA template (formally described as a DNA-dependent RNA polymerase).

FIGURE :Promoters and terminators define the unit


Transcription dr harinikumar k m university of agricultural sciences bengaluru karnataka

  • Promoter –A region of DNA where RNA polymerase binds to initiate transcription.

  • Start point –The position on DNA corresponding to the first base incorporated into RNA.

  • Terminator –A sequence of DNA that causes RNA polymerase to terminate transcription.

  • Transcription unit – The sequence between sites of initiation and termination by RNA polymerase; it may include more than one gene.


Transcription dr harinikumar k m university of agricultural sciences bengaluru karnataka

  • Upstream –Sequences in the opposite direction from expression.

  • Downstream –Sequences proceeding farther in the direction of expression within the transcription unit.

  • Primary transcript –The original unmodified RNA product corresponding to a transcription unit.


Types of rnas produced in cells

Types of RNAs Produced in Cells


Phases of transcription

Phases of Transcription

  • Initiation:Binding of RNA polymerase to promoter, unwinding of DNA, formation of primer.

  • Elongation:RNA polymerase catalyzes the processive elongation of RNA chain, while unwinding and rewinding DNA strand

  • Termination:termination of transcription and disassemble of transcription complex.


Rna polymerase structure theme and variation on five subunits

RNA Polymerase Structure: Theme and Variation on Five Subunits


Four rna polymerases of eukaryotic cells

Four RNA Polymerases of Eukaryotic Cells


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Initiation RNA pol I RNA pol III RNA pol II

__________________________________________________

ATP requirement no no yes

__________________________________________________

A and B or TATA box

core consensus sq. core element C box Inr

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CAAT box

upstream element UCE GC box etc

__________________________________________________

general TFs SL1 TFIIIA B C various TFIIs

___________________________________________________

upstream factors UBF various up-

stream factors

_____________________________________________________


Transcription initiation by rna polymerase ii

Transcription Initiation by RNA Polymerase II

  • Initiation occurs at an initiation site at the 3’ end of the gene. The initiation site is part of a larger promoter. Each gene has its own promoter. The promoter consists of a TATA box of about 100 nucleotides, mostly T and A, and an initiation sequence. The TATA box is upstream of the initiation sequence.

  • Proteinaceous transcription factors attach to the promoter and help the polymerase find and attach to the initiation site. RNA polymerase attaches to the initiation sequence.


Bacterial rna polymerase consists of multiple subunits

Bacterial RNA Polymerase Consists of Multiple Subunits

  • Holoenzyme – The RNA polymerase form that is competent to initiate transcription. It consists of the five subunits of the core enzyme and σ factor.

  • Bacterial RNA core polymerases are ~400 kDmultisubunit complexes with the general structure α2ββ′. Catalysis derives from the β and β′ subunits.

  • CTD (C-terminal domain) – The domain of RNA polymerase that is involved in stimulating transcription by contact with regulatory proteins.

FIGURE : RNA polymerase has 4 types of subunit


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RNA-pol of E. Coli

Rifampicin, a therapeutic drug for tuberculosis treatment, can bind specifically to the  subunit of RNA-pol, and inhibit the RNA synthesis.


E coli rna polymerase

E. Coli RNA Polymerase

  • Bacterial RNA polymerase can be divided into the α2ββ′ core enzyme that catalyzes transcription and the  subunit that is required only for initiation.

  • The β’ subunit is involved in DNA binding.

  • The β subunit contains the polymerase active site.

  • The α subunit acts as scaffold on which the other subunits assemble.

Site of DNA binding and RNA polymerization


Sigma factor

Sigma factor

  • Also requires sigma factor for initiation –forms holo enzyme complex

  • Sigma factor changes the DNA-binding properties of RNA polymerase so that its affinity for general DNA is reduced and its affinity for promoters is increased.

  • The Sigma factor is required for binding of the RNA polymerase to the promoter

  • Association of the RNA polynerase core complex omega the Sigma factor forms the holo-RNA polymerase complex

  • omega the Sigma factor the core complex binds to DNA non-specifically.

FIGURE : Sigma factor controls specificity


Sigma factors

Sigma factors

  • decreases the affinity of the RNA polymerase to non-promoter regions

  • Different Sigma factors for specific classes of genes

  • Different sigma factors bind RNAP and recognize specific -10 ,-35 sequences

  • Helps melt DNA to expose transcriptional start site

  • Most bacteria have major and alternate sigma factors

  • Promote broad changes in gene expression

    • E. coli 7 sigma factors

    • B. subtilis 18 sigma factors

      Generally, bacteria that live in more varied environments have more sigma factors


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Sigma factors

s70

s54

sS

sS

sF

s32

Extreme heat shock, unfolded proteins

E. coli can choose between 7 sigma factors and about 350 transcription factors to fine tune its transcriptional output


The holoenzyme goes through transitions in the process of recognizing and escaping from promoters

The Holoenzyme Goes through Transitions in the Process of Recognizing and Escaping from Promoters

  • When RNA polymerase binds to a promoter, it separates the DNA strands to form a transcription bubble and incorporates nucleotides into RNA.

FIGURE :- RNA polymerase surrounds the bubble


Transcription occurs by base pairing in a bubble of unpaired dna

Transcription Occurs by Base Pairing in a “Bubble” of Unpaired DNA

  • RNA polymerase separates the two strands of DNA in a transient “bubble” and uses one strand as a template to direct synthesis of a complementary sequence of RNA.

  • The bubble is 12 to 14 bp, and the RNA–DNA hybrid within the bubble is 8 to 9 bp.

  • RNA-polrecognizesthe TTGACA region, and slides to the TATAAT region, then opens the DNA duplex.

  • The unwound region is about 171bp.

  • No primer is needed for RNA synthesis

FIGURE RNA synthesis occurs in the transcription bubble


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FIGURE :- RNA polymerase passes through several steps prior to elongation


Transcription dr harinikumar k m university of agricultural sciences bengaluru karnataka

  • Ternary complex – The complex in initiation of transcription that consists of RNA polymerase and DNA as well as a dinucleotide that represents the first two bases in the RNA product.

  • There may be a cycle of abortive initiations before the enzyme moves to the next phase.

  • Sigma factor is usually released from RNA polymerase when the nascent RNA chain reaches ~10 bases in length.


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FIGURE:- RNA polymerase changes size at initiation


Sigma factor controls binding to dna by recognizing specific sequences in promoters

Sigma Factor Controls Binding to DNA by Recognizing Specific Sequences in Promoters

  • conserved sequence – Sequences in which many examples of a particular nucleic acid or protein are compared and the same individual bases or amino acids are always found at particular locations.

  • A promoter is defined by the presence of short consensus sequences at specific locations.

  • The promoter consensus sequences usually consist of a purine at the startpoint, a hexamer with a sequence close to TATAAT centered at ~ –10(–10 element or TATA box), and another hexamer with a sequence similar to TTGACA centered at ~–35(–35 element).


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  • Individual promoters usually differ from the consensus at one or more positions.

  • Promoter efficiency can be affected by additional elements as well.

  • UP element – A sequence in bacteria adjacent to the promoter, upstream of the –35 element, that enhances transcription.

FIGURE 14: DNA elements and RNA polymerase modules contributing to promoter recognition by sigma factor


Promoter efficiencies can be increased or decreased by mutation

Promoter Efficiencies Can Be Increased or Decreased by Mutation

  • Down mutationsto decrease promoter efficiency usually decrease conformance to the consensus sequences, whereas up mutations have the opposite effect.

  • Mutations in the –35 sequence can affect initial binding of RNA polymerase.

  • Mutations in the –10 sequence can affect binding or the melting reaction that converts a closed to an open complex.


Transcription factors

Transcription Factors

  • TFAIIA, TFAIIB – components of RNA polymerase II holo-enzyme complex

  • TFIID – Initiation factor, contains TATA binding protein (TBP) subunit. TATA box recognition.

  • TFIIF – (RAP30/74) decrease affinity to non-promoter DNA


Class ii transcription factors

Class II transcription factors

  • TFIIA activates TBP by relieving the repression that is caused by the TAFs

  • TFIIB binds adjacent to TBP and TATA box

  • TFIID is a complex protein containing a TATA-box binding protein and 8-10 TBP-associated factors (TAFs)

    • TBP:TATA-binding protein

    • TAFs:TBP-associated factors

  • TFIIF consists of two subunits. The larger subunit has an ATP-dependent DNA helicase activity and the small one contacts the core polymerase.

  • TFIIE and TFIIH are required for promoter clearance to allow RNA polymerase to commence movement away from the promoter.


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TF for eukaryotic transcription


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polⅡ

TFⅡF

TFⅡH

TAF

TAF

TAF

TFⅡA

TBP

TFⅡB

DNA

TATA

RNA polⅡ with transcription factors form transcription initiation complex. TF II D is the only factor which can recognize specific sites.


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POL-Ⅱ

POL-Ⅱ

TFⅡF

CTD-

P

Pre initiation complex

ⅡB

TBP

TBP

TBP

TBP

TAF

TAF

TAF

TATA

ⅡA

TFⅡD-ⅡA-ⅡB-DNA complex

CTD(Carboxyl Terminal Domain ) is repeated sequence of Tyr-Ser-Pro-Thr-Ser-Pro-Ser

ⅡH

ⅡE

ⅡH

ⅡE

TFⅡF

TBP

TAF

ⅡB

TATA

ⅡA

CTD tail of RNA pol II is phosphorylated by TFⅡH


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Pre-initiation complex (PIC)

  • TBP of TFII D binds TATA

  • TFII A and TFII B bind TFII D

  • TFII F-RNA-pol complex binds TFII B

  • TFII F and TFII Eopen the dsDNA (helicase and ATPase)

  • TFII H: completion of PIC


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Model of RNA PolII Preinitiation Complex


Interactions between sigma factor and core rna polymerase change during promoter escape

Interactions between Sigma Factor and Core RNA Polymerase Change During Promoter Escape

  • A domain in sigma occupies the RNA exit channel and must be displaced to accommodate RNA synthesis.

  • Abortive initiations usually occur before the enzyme forms a true elongation complex.

  • Sigma factor is usually released from RNA polymerase by the time the nascent RNA chain reaches ~10 nt in length.

FIGURE :-Sigma and core enzyme must dissociate


Enzyme movement

Enzyme Movement

  • DNA moves through a channel in RNA polymerase and makes a sharp turn at the active site.

  • Changes in the conformations of certain flexible modules within the enzyme control the entry of nucleotides to the active site.

FIGURE :- DNA turns at the active site


Elongation

Elongation:

  • RNA polymerase moves along, unwinding one turn of the double helix at a time thus exposing about 10 bases.

  • New RNA nucleotides are added to the 3’ end of the growing mRNA molecule at a rate of about 60 sec-1.

  • The double helix reforms behind the enzyme. Many RNA polymerase molecules can transcribe simultaneously (remember, the gene is hundreds of thousands of nucleotides long).

  • Only one DNA strand, the coding strand, is transcribed. The other strand is not used (silent). But, which strand is coding and which silent varies from gene to gene.

  • The reading direction is 3’ to 5’ along the coding strand.


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  • Polymerase is accurate - only about 1 error in 10,000 bases

  • Even this error rate is OK, since many transcripts are made from each gene

  • Elongation rate is 20-50 bases per second - slower in G/C-rich regions

  • Topoisomerases precede and follow polymerase to relieve supercoiling


Termination

Termination:

  • Process by which RNA polymerase complex disassembles from 3’ end of gene.

  • There are two classes of terminators: Those recognized solely by RNA polymerase itself without the requirement for any cellular factors are usually referred to as “intrinsic terminators.”

    • Others require a cellular protein called rho and are referred to as “rho-dependent terminators.”


Transcription dr harinikumar k m university of agricultural sciences bengaluru karnataka

1. Rho-independent termination

  • Termination sequence has 2 features:

    • Series of U residues

    • GC-rich self-complimenting region

  • GC-rich sequences bind forming stem-loop

  • Stem-loop causes RNAP to pause

  • U residues unstable, permit release of RNA chain


2 rho independent termination

2. Rho-independent termination

  • Rho factor is a protein that binds to nascent RNA and tracks along the RNA to interact with RNA polymerase and release it from the elongation complex.

  • rut – An acronym for rho utilization site, the sequence of RNA that is recognized by the rho termination factor.

  • Rho is hexameric protein

  • 70-80 base segment of RNA wraps around

  • Rho has ATPase activity, moves along RNA until site of RNAP, unwinds DNA/RNA hybrid

  • Termination seems to depend on Rho’s ability to “catch up” to RNAP

  • No obvious sequence similarities, relatively rare


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FIGURE :- Rho terminates transcription


How does rho factor work

How Does Rho Factor Work?

  • polarity – The effect of a mutation in one gene in influencing the expression (at transcription or translation) of subsequent genes in the same transcription unit.

  • antitermination complex – Proteins that allow RNA polymerase to transcribe through certain terminator sites.

FIGURE :- Rho can terminate when a nonsense mutation removes ribosomes


Supercoiling is an important feature of transcription

Supercoiling Is an Important Feature of Transcription

  • Negative supercoiling increases the efficiency of some promoters by assisting the melting reaction.

  • Transcription generates positive supercoils ahead of the enzyme and negative supercoils behind it, and these must be removed by gyrase and topoisomerase.

FIGURE :- Transcription changes DNA structure


Competition for sigma factors can regulate initiation

Competition for Sigma Factors Can Regulate Initiation

  • E. coli has seven sigma factors, each of which causes RNA polymerase to initiate at a set of promoters defined by specific –35 and –10 sequences.

FIGURE :- Sigma controls promoter recognition


Competition for sigma factors can regulate initiation1

Competition for Sigma Factors Can Regulate Initiation

  • The activities of the different sigma factors are regulated by different mechanisms.

  • anti-sigma factor – A protein that binds to a sigma factor to inhibit its ability to utilize specific promoters.

FIGURE :- E. coli has several sigma factors


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