Moderator : Dr Smita S SonoliSpeaker: Mohit Adhikary

Outline • Introduction• Prokaryotic transcription• Eukaryotic transcription• Post transcriptional modification

50%

25%15%

10%

Difference between replication &transcription

REPLICATION TRANSCRIPTION• New DNA is formed• DNA-DNA hybrid

complex• DNA polymerase enz.• Primer is requried• Deoxyrinonucleotides

used• Entire genome is

copied.• Proofreading• Genetic information is

inherited.

• New RNA is formed• DNA –RNA hybrid

complex• RNA polymerase enz.• Primer not requrired• Ribonucleotides used

• Very small portion of genome transcribed

• No proofreading.• Information is

transferred from gene to protein.

4 types of RNA

• mRNA• rRNA• tRNA• smallRNA– snRNA– scRNA

Central dogma of molecular biology

Replication

GENOME

TRANSCRIPTOME

PROTEOME

Transcription

Translation

CURRENT CONCEPT

Replication, transcription and translation

• The process in a cell by which genetic material is copied from a strand of DNA to a complementary strand of RNA.

OR The synthesis of RNA from a DNA template.• TWO aspects of transcription must be

considered• The enzymology• The signal that determine where on a

DNA molecule transcription begins and stops.

TRANSCRIPTION

Transcription

RNA polymerase

σα

β

β'

Multiple functions of RNA polymerase

• Searches promoter sites• Unwinds DNA helical• Selection of correct ribonucleoside

triphosphate• Detects termination signals• Interacts activator & repressor proteins• Essential function of RNAP in all cells make

them attractive targets for antibiotics & other drugs.

Types of RNAP• Type I or A:– rRNA– Not inhibited by amantin

• Type II or B: – main enzyme synthesizing mRNA– Inhibited by amanitin– snRNA & miRNA – Has 2 large and 12 smaller sub units– Activated by phosphorylation

• Type III or C: – tRNA– Moderately sensitive to amanitin

Difference between DNA & RNA Polymerase

• RNA P• No primer

required• Does not posses

endo & exonuclease activity

• No proof reading

• DNA P• Primer

required• Posses endo &

exonuclease activity• Proof reading

done

Prerequisite of transcriptionProkaryote • Substrate

– NTPs• ATP• GTP • CTP• UTP

• DNA dependent RNA Polymerase (2α 1β1β’ 1δ) with 2 Zn molecule

Eukaryote • Substrate

– NTPs• ATP• GTP • CTP• UTP

• RNAP type I / A – rRNA (large)RNAP type II / B – mRNA & snRNARNAP type III / C – tRNA & small rRNA

Transcription unit

Steps in transcription

-355’ TTGACA-3’

-105’ TATAAT-3’Pribnow box

+1Initiation

Cyclic AMP receptor protein (CRP)

3’ end 3’ end

3’ end 5’ end

PPi P

PiPPi P

PiPPi

PPi

PPi P

Pi

PPi

PPiP

Pi

PPiPPi

NTPs

pause

Termination

• Two types– Rho dependent

termination– Rho independent

termination

3’ end

5’ end 5’ end

Rho dependent termination

Rho independent termination

• Formation of hair pins of newly synthesised RNA.• RNA self

complementary

“N” represents a non-complementary base.

Action of antibiotics

• Rifampicin

• Dactinomyci

n

Transcription in Eukaryotes

• Separate polymerases for synthesis

of rRNA, tRNA, mRNA

• Transcription factors

Chromatin structure & Gene expression

• Association of DNA with histones• Acetylation of lysine residues at amino

terminus of histone protein• Histone acetyltransferase

• Histone deacetylase

Nuclear RNA polymerase of eukaryotic cells

• RNA Polymerase I– Synthesizes the precursor of 28S, 18S &

5.8S rRNA• RNA Polymerase II• RNA Polymerase III• Synthesizes tRNA, 5SrRNA, snRNA & snoRNA

• Mitochondrial RNA polymerase

RNA Polymerase IIPromoters and transcriptional factors for RNA synthesis• TATA or Hogness box• CAAT box GC rich box• TFs • TFIID: recognises and binds the TATA box• TFFIF: brings polymerase to the promoter• TFIIH:• Helicase: melts DNA• Kinase: phosphorylates polymerase

Role of enhancer in gene regulation

• Cis acting DNA sequence: inc. rate• +nt on the same chromosome• They can– Be located upstream or down stream of

transcription start site– Be close or thousands of base pair away

from the promoter– Occur on either strand of DNA

Post- transcriptional modification.

DNA

Primary transcript (inactive)

Functionally active RNA molecules

transcription

Alteration (splicing, base modification)

Ribosomal RNA• Synthesised by long precursor pre-ribosomal RNA• Prokaryotes 23s,16s,5s• Eukaryotes 28s,18s,5.8s

•Later trimmed by exonucleases•Modified at some bases & ribose

Transfer RNA

• An intron removed from anticodon loop by

nucleases

• Seq. of 3’ 5’ must be trimmed

• Addition of CCA by nucleotidyltransferase

• Modification of bases at specific position

(dihydrouracil)

Before After

Messenger RNAPrimary transcript subjected to:

• 5’ capping

• Poly A tail

• Removal of introns

• splicing

5’ capping

Guanine7-methyltransferase

Poly A tail

Introns

Exons

Systemic lupus erythematosus, an

often fatal inflammatory disease,

results from an autoimmune response

in which the patient produces

antibodies against host proteins,

including snRNP.

Effect of splice site mutations: Mutations at splice sites can lead to improper splicing and the production of aberrant proteins. It is estimated that fifteen percent of all genetic diseases are a result of mutations that affect RNA splicing. For example, mutations that cause the incorrect splicing of β-globin mRNA are responsible for some cases of β-thalassemia—a disease in which the production of the β-globin protein is defective

Alternative splicing patterns in eukaryotic mRNA.

DNA

Cytoplasm

Nucleus

Summary

ExportG AAAAAA

RNATranscription

Nuclear pores

G AAAAAA

RNAProcessing

mRNA

Reverse transcription

RNA dependent DNA polymerase.

retro viruses carry RNA as their genetic material & can synthesize double stranded DNA from their genomic RNA by process know Reverse transcription.

Viral DNAP is called reverse transcriptase .

Exa. HIV

• Viral DNAP is called reverse transcriptase.

• RNA dependent DNA polymerase.

• Retroviruses carry RNA as their genetic material & can synthesize double stranded DNA from their genomic RNA by process REVERSE TRANSCRIPTION.

• Eg. HIV

“We don’t see things as they are, we see things as we are.”

-Anais Nin