DNA TRANSCRIPTION

Rikesh Lal Shrestha

M.Pharm (2015), Industrial

Kathmandu University

Contents• INTRODUCTION

• DNA TRANSCRIPTION STAGES

• POST TRANSCRIPTION MODIFICATION

5’ CAPPING

POLY (A) TAIL

SPLICING

• REVERSE TRANSCRIPTION

• DRUGS INHIBITING TRANSCRIPTION

Introduction• The synthesis of an RNA molecule from DNA molecule in the

presence of enzyme RNA polymerase is called Transcription.

• Information stored in DNA is transferred to RNA moleculesduring transcription.

• All eukaryotic cells have five major classes of RNA:ribosomal RNA (rRNA), messenger RNA (mRNA), transferRNA (tRNA), small nuclear RNA and microRNA (snRNA andmiRNA).

• The first three are involved in protein synthesis, while thesmall RNAs are involved in mRNA splicing and regulation ofgene expression.

Similarities between Replication and Transcription

The processes of DNA and RNAsynthesis are similar in thatthey involve-

(1) The general steps ofinitiation, elongation, andtermination with 5' to 3'polarity;

(2) Large, multicomponentinitiation complexes; and

(3) Adherence to Watson-Crickbase-pairing rules.

Replication Transcription

DNA replication is catalyzed by DNA

polymerase which needs a primer.

RNA synthesis is catalyzed by RNA

polymerase

Deoxyribonucleotides are used in DNA

synthesis

Ribonucleotides are used in RNA

synthesis

Thiamine is the complementary base pair

for Adenine in DNA.

Uracil is the complementary base pair for

Adenine in RNA.

The entire genome must be copied during

DNA replication

Only a portion of the genome is

transcribed or copied into RNA

There is proofreading function during

DNA replication.

There is no proofreading function during

RNA transcription.

Differences between Replication and Transcription

Template strand•The DNA strand that is transcribed or copied into an RNA molecule is called template strand.

•The another non-template DNA strand, is called coding strand.

•The information in the template strand is read out in the 3' to 5' direction

•The information in the RNA molecule is read out in the 5' to 3' direction

•In the coding strand the sequence is same as that of the sequence of RNA primary transcript. With the exception of T for U changes.

Transcription unit• A transcription unit is defined as that region of DNA that includes the signals for

transcription initiation, elongation, and termination.

• The nucleotide in the promoter adjacent to the transcription initiation site is designated -1,

• These negative numbers increase as the sequence proceeds upstream, away from the

initiation site

• The nucleotide in the initiation site is designated +1,

• These positive numbers increase as the sequence proceeds downstream, away from the

initiation site

Bacterial DNA-Dependent RNA Polymerase

The DNA-dependent RNA polymerase (RNAP) of the bacterium Escherichia coli exists as an approximately 400 kDa core complex consisting of-

•two identical α subunits,

•similar but not identical β and β ' subunits, and

•an ω subunit and a

•A sigma subunit (σ)

•Beta is thought to be the catalytic subunit.

Bacterial DNA-Dependent RNA Polymerase

• RNAP, a metalloenzyme, also contains two zinc molecules.

• The core RNA polymerase associates with a specific protein factor (the sigma σ factor) that helps the core enzyme recognize and bind to the specific deoxynucleotide sequence of the promoter region to form the preinitiationcomplex (PIC)

• Bacteria contain multiple factors, each of which acts as a regulatory protein.

Mammalian DNA-Dependent RNA

Polymerases

Mammalian cells possess three distinct nuclear

DNA-Dependent RNA Polymerases

• RNA polymerase I is for the synthesis of

rRNA

• RNA polymerase II is for the synthesis of

mRNA and miRNA

• RNA polymerase III is for the synthesis of

tRNA/5S rRNA, snRNA

Prokaryotic versus Eukaryotic TranscriptionParameters Prokaryotic Cell Eukaryotic Cell

Location Transcription Occur in cytoplasm of Prokaryotic cell

Transcription Occur in nucleus of Eukaryotic cell

RNA Polymerase

Single RNA polymerase have capability to produce rRNA, mRNA & tRNA.

Three classes of RNA polymerases (I, II,III). RNA Poly. I rRNA, RNA Poly.II

mRNA, RNA Poly.III tRNA

Synthesized RNA

The RNA produce is fully functional for translation process.

The RNA produced is not fully functional and k/a premature m-RNA which need to be modified to produce mature m-RNA that can synthesize protein.

Process Transcription along with translation process take place. So the process is also k/a transcription coupled translation.

After transcription complete then only translation process take place.

TranscribedRNA

Transcribed RNA is polycistronic (i.e multiple genes are regulated by only one promoter) . So, multiple proteins are produce in chain.

Transcribed RNA is monocistronic (i.esingle gene is placed on single promoter)

Stages of Transcription

Both prokaryote and eukaryote have 3 stage of transcription. They are:

• Initiation

• Elongation

• Termination

Initiation in Prokaryote• The RNA polymerase and sigma factor combine to form a molecule

called holoenzyme.

• This holoenzyme binds to the promoter region of double strands DNAforming closed complex.

• Then holoenzyme unwind DNA helix , thus open promoter complex isformed.

• After 10–20 nucleotides have been polymerized, holoenzyme undergoesconformational change leading to promoter clearance.

• Once this transition occurs, holoenzyme moves away from the promoter,transcribing down the transcription unit, leading to the next phase of theprocess, elongation.

Elongation in Prokaryote• As the elongation complex that is holoenzyme progresses along the DNA

strands, it unwinding DNA to provide access for the complementary basepairing to the nucleotides of the template strand.

• After addition of few riboneucleotides the sigma factor dissociate fromholoenzyme . Then RNA polymerase adds nucleotides to 3’ –OH group.Thus elongation proceed in the direction of core enzyme.

Termination in Prokaryote• Two types of terminator sequences

occur in prokaryotes:

•Rho() Independent: In RNA sequencethere occurs G and C rich region, theirPalindromic repeat forms a hairpin loop.Beyond the hair pin, RNA sequencecontains a strings of Us, the bonding ofUs to the corresponding As is weak. Thisfacilitates the dissociation of the RNAfrom DNA.

•Rho() dependent: A sequence of DNAtemplate strand signal is recognized byprotein k/a Rho () factor proteins, itattach and climb RNA strand then breakthe hydrogen bonds between thetemplate DNA and RNA .

Initiation in EukaryoteWe can divide eukaryotes promoter into two regions:

The core promoters elements. The best characterized are TATA Box = TATAAAA, located at about position -30 bp(*AT-rich DNA is easier to denature than GC-rich DNA)

Promoter proximal elements (located upstream, ~-50 to -200 bp) “Cat Box” = CAAT and “GC Box” GGGCGG

General Transcription factors (GTF) are proteins that help eukaryotic RNA polymerase recognize promoter sequences.

Binding of GTFs and RNA polymerase occurs in set of order.

Complete complex (RNA polymerase + GTFs) is called a pre-initiation complex(PIC).

Transcription regulatory proteins (activators) bind to the enhancers region in ds DNA and also to RNA polymerase II in order to increase the rate of transcription initiation of eukaryotic genes.

Initiation in EukaryoteOrder of binding is: IID + IIA + IIB + RNA poly. II + IIF +IIE +IIH

Elongation in Eukaryote• As RNA polymerase moves along the DNA it continues to

untwist the double helix, exposing about 10 to 20 DNA basesat a time for pairing with RNA nucleotides.

• RNA polymerase synthesizes a single strand of RNA againstthe DNA template strand (anti-sense strand), addingnucleotides to the 3’ end of the RNA chain.

Elongation

RNA

polymerase

Non-template

strand of DNARNA nucleotides

3 end

A E G C A

U

T A G G T T

AT C C A A

3

5

5

Newly made

RNA

Direction of transcription

(“downstream”) Template

strand of DNA

Termination in Eukaryote• Specific sequences in the DNA signal termination of

transcription (AAUAAA)

• When one of these is encountered by the polymerase, theRNA transcript is released from the DNA and the doublehelix can zip up again.

POST TRANSCRIPTION MODIFICATION5’ capping

• It is a 7-methylguanosine tri-phosphate cap structure at the5' terminal of eukaryotic mRNA.

• The cap structure is added to the 5' end of the newlytranscribed mRNA before it transport to cytoplasm.

• The 5' cap of the RNA transcript is required both for efficienttranslation initiation and protection of the 5' end of mRNAfrom attack by 5-'3' exonucleases.

5’ capping•There is addition of the Guanosine triphosphate to 5’terminal of mRNAby enzyme guanylyl transferase.

•The 5’-end of the mRNA is capped 5’ to 5’ with a guanine nucleotideforming triphosphate bridge.

•Methylation occurs in N7 of guanine by guanine-7-methyl transferase.

•Additional methylation steps may occur.

•The secondary methylations of mRNA molecules, those on the 2'-hydroxy and the N6 of base residues, occur after the mRNA molecule hasappeared in the cytoplasm.

Poly (A) tail• Poly(A) tails are added to the 3' end of mRNA molecules in a posttranscriptional

processing step.

• The mRNA is first cleaved about 20 nucleotides downstream from an AAUAAA

recognition sequence.

• Another enzyme, poly(A) polymerase adds a poly(A) tail which is subsequently

extended to as many as 200 A residues.

• The poly(A) tail appears to protect the 3' end of mRNA from 3' -5' exonuclease

attack.

RNA Splicing• Splicing : Removal of introns

• Introns: non-coding sequences

between exons

• Exons: amino acid coding sequences

are spliced.

• “snurps” snRNP bind to splice site and

form a spliceosome

• Spliceosome excises the intron and

rejoins the exons.

• Mature mRNA is produce that moves

into cytosol from nuclear pore for

protein synthesis.

Reverse Transcription• It is the process of synthesis double stranded

DNA from Single stranded RNA by reverse

transcriptase enzyme (RNA directed DNA

polymerase).

• Reverse transcriptase common in HIV,

MMLV(Moloney Murine Leukemia Virus),

AMV(Avian Myeloblastosis Virus)

• Reverse transcriptase enzyme includes two

activity: DNA polymerase and RNAase H

Retrovirus Cycle

Drug Inhibiting Transcription• Rifampicin binds with Beta subunit of

prokaryotic RNA polymerase but not toeukaryotic RNA polymerases. Rifampicin usefor the treatment of tuberculosis and leprosy.

• Mitomycin used as anticancer drug Intercalates with DNA strands blocks transcription.

• Alpha amanitin is a molecule made from the“death cap” mushroom and is a known potentinhibitor RNA polymerase. The mechanism ofaction is that alpha amanitin inhibits RNApolymerase –II at both the initiation andelongation states of transcription.

• Actinomycin D- Intercalates with DNA strands .Actinomycins inhibit both DNA synthesis and RNA synthesis by blocking chain elongation. Actinomycins are used as anticancer drugs

REFERENCE• Professor (Dr.) Namrata Chhabra Biochemistry For Medics-

Lecture Notes

• Chapter 12 of Molecular Biology of the Gene 6th Edition(2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine,M, Losick, R. 377-414.

• Murakami KS, Darst SA. (2003) Bacterial RNA polymerases:the wholo story. Curr Opin Struct Biol 13:31-9.

• Campbell, E, Westblade, L, Darst, S., (2008) Regulation ofbacterial RNA polymerase factor activity: a structuralperspective. Current Opinion in Micro. 11:121-127

• John Wiley & Sons, Inc, Transcription and RNA Processing

• Biochemistry For Medics- Lecture Notes.

THANK YOU……!!!!