Transcription in prokaryotes

In prokaryotes, only one RNA polymerase is responsible for the synthesis of all RNA (except for the RNA primers required for replication)

Compare the mechanism of action of RNA polymerase and DNA polymerase.

[1][1]

RNA POLYMERASE

’

Core Enzyme2, ’ and

Holoenzyme2, ’ and and

Similarities• Substrate for both - nucleoside triphosphates• Pyrophosphate released for every NTP added• Always in the 5’3’ directionDifferences• Recognizes the site for initiation of

transcription• Ribose sugar and uracil• No primer required• No proofreading activity

[2][2]For genes to be transcribed by RNA

polymerase the appropriate initiation site must be recognized.

I.How does the RNA polymerase know where to start transcription?II.How does the sequence of the

Pribnow box relate to its function?

PROMOTERS

+1

TATAAT box

TTGACA

Transcription start site

~7 bases~19 bases

5’

3’

3’

5’

Pribnow box-35 sequence

Each of the two DNA strands can serve as templates for transcription

I. What determines which of the two DNA strands will be transcribed??

II. Can both the DNA strands be transcribed??

III. Which is the sense (coding) and anti- sense (noncoding) strand ??

IV. The RNA has the same sequence as which one of the two DNA strands?

[3][3]

What determines which of the two DNA strands will be transcribed??the location of the promoter

Can both the DNA strands be transcribed??Yes, but for a given gene only one strand will be the template

Which is the sense (coding) and anti- sense (noncoding) strand ??Sense strand = the same sequence as the mRNA, contains the promoterantisense = acts as the template, complementary sequence to the mRNA.

[3][3]

Transcription

[4][4]Three separate stages of transcription

INITIATION, ELONGATION, TERMINATION

TERMINATIONindependent

TERMINATION - independent

TERMINATION - independent

Action Of Antibiotics on Transcription

• Rifampin - binds to subunit of prokaryotic RNA polymerase– tuberculosis

• Dactinomycin – binds to DNA, prevents the movement of RNA polymerase– Tumor chemotherapy

TRANSCRIPTION -TRANSCRIPTION -EUKARYOTESEUKARYOTES

RNA Polymerases in Eukaryotes

RNA POLYMERASE I - rRNA

RNA POLYMERASE II - mRNA, snRNAs

RNA POLYMERASE III - tRNA and other small RNAs

Eukaryotic gene Eukaryotic gene structurestructure

Exon 1 Exon 4Exon 3Exon 2 Exon 5

PROTEIN

5’ 3’

= intron (untranslated)

= exon (translated)

Pre-mRNA

Exon 1 Exon 4Exon 3Exon 2 Exon 5mRNA

RNA Polymerase II• 10-12 subunits• The largest subunit contains a C-

terminal region or domain (CTD) with 52 repeats of the amino acid sequence PTSPSYS

T = threonineS = serineY = tyrosine

Can be phosphorylated

RNA PLOYMERSE II PROMOTERS

+1-20-30

TATA box

CAAT box

Transcription start site

25 bases45-55 bases

-70

5’

3’

3’

5’

Transcription factorsTranscription factors• Any protein that regulates transcription but is not

a part of RNA polymerase, is a transcription factor• Six general transcription factors (GTFs) required

for all promoters

– TFIIA– TFIIB– TFIID– TFIIE– TFIIF– TFIIH

+ RNA Polymerase II = Preinitiation complex

TATA box

TFIID BA

Pol IIF

E H

Complex of several proteins, TATA-Binding Protein (TBP) and ~12 TBP Associated Factors (TAFs)

TFIIDA TFIIA stabilizes the DNA and TFIID interactionB TFIIB binds to TFIID and acts as bridge between Pol II and TFIID

F TFIIF binds Pol II and targets its to the promoterPREINITIATION COMPLEX (PIC)PREINITIATION COMPLEX (PIC)E Recruits TFIIHH Promoter melting, kinase activity

TFIID BA

E HPol IIF

TFIID BA

E HPol IIF

mRNA

GTFs only produce a basal level of transcription

Enhancers/Silencers• Upstream or downstream• Close to the promoter or thousands of base

pairs away• On either of the two strands of DNA• Act through intermediary or gene specific

transcription factors proteins• Enhancers activate transcription • Silencers deactivate transcription

Enhancers/Silencers

Response Elements•Enhancers contain response elements that

are responsive to certain metabolic factors

–Cyclic AMP response element (CRE)–Glucocorticoid response element (GRE)–Heat shock element (HSE)

•REs bind transcription factors produced under certain cell conditions to activate several related genes

-amanitin and RNA Pol II

Post Transcriptional Post Transcriptional Modification – mRNAsModification – mRNAs

5’ Capping

Post Transcriptional Post Transcriptional Modification – mRNAsModification – mRNAs

Poly-A tail

Post Transcriptional Post Transcriptional Modification – mRNAsModification – mRNAs

SPLICING

Post Transcriptional Post Transcriptional Modification - rRNAsModification - rRNAs

RNA Polymerase I

Post Transcriptional Post Transcriptional Modification - tRNAsModification - tRNAs

THE END!