Basic principle of transcription

by: Dr. Alsadig Gassoum

Assistant professor Al-Madain College for medical Sciences and

technologyACMST

TranscriptionTranscription is the synthesis of a single stranded

RNA from a double stranded DNA template. RNA synthesis occurs in the 5→3 direction and its

sequence corresponds to that of the DNA strand which is known as the sense strand

Transcription

• RNA Polymerase– Reads DNA• Makes RNA copy of

– One strand– One gene

Transcriptiono is the enzymic synthesis of RNA on a DNA template

o is the first stage in the overall process of gene expression

o Transcription is catalyzed by an RNA polymerase

o requires a dsDNA template as well as the precursor ribonucleotides ATP, GTP, CTP and UTP

Transcriptiono RNA synthesis always occurs in a fixed direction,

from the 5- to the 3-end of the RNA molecule

o only one of the two strands of DNA becomes transcribed into RNA.

o One strand is known as the sense strand

Transcriptiono The sequence of the RNA is a direct copy of the

sequence of the deoxynucleotides in the sense strand (with U in place of T).

o The other strand is known as the antisense strand

InitiationRNA polymerase is the enzyme responsible for

transcription.

It binds to specific DNA sequences called promoters to initiate RNA synthesis.

These sequences are upstream (to the 5-end) of the region that codes for protein, and they contain short, conserved DNA sequences which are common to different promoters.

InitiationThe RNA polymerase binds to the dsDNA at a

promoter sequence, resulting in local DNA unwinding.

The position of the first synthesized base of the RNA is called the start site and is designated as position +1.

Initiationinvolves the binding of an RNA polymerase to the

dsDNA.

RNA polymerases are usually multisubunit enzymes

They bind to the dsDNA and initiate transcription at sites called promoters

InitiationPromoters are sequences of DNA at the start of genes,

that is to the 5-side (upstream) of the coding region.

Sequence elements of promoters are often conserved between different genes.

InitiationThe short conserved sequences within promoters are

the sites at which the polymerase or other DNA-binding proteins bind to initiate or regulate transcription.

The DNA helix must be locally unwound.

Unwinding begins at the promoter site to which the RNA polymerase binds.

InitiationThe polymerase then initiates the synthesis of the

RNA strand at a specific nucleotide called the start site (initiation site).

This is defined as position +1 of the gene sequence

Initiation

The RNA polymerase and its co-factors, when assembled on the DNA template, are often referred to as the transcription complex.

ElongationRNA polymerase moves along the DNA and

sequentially synthesizes the RNA chain.

DNA is unwound ahead of the moving polymerase, and the helix is reformed behind it.

ElongationThe RNA polymerase covalently adds ribonucleotides

to the 3-end of the growing RNA chain

The polymerase therefore extends the growing RNA chain in a 5→3 direction.

This occurs while the enzyme itself moves ina 3→5 direction along the antisense DNA strand

(template).

Elongation

As the enzyme moves, it locally unwinds the DNA, separating the DNA strands, to expose the template strand for ribonucleotide base pairing and covalent addition to the 3-end of the growing RNA chain.

ElongationThe helix is reformed behind the polymerase.

The E. coli RNA polymerase performs this reaction at a rate of around 40 bases per second at 37°C.

Termination RNA polymerase recognizes the terminator which

causes no further ribonucleotides to be incorporated. This sequence is commonly a hairpin structure. Some terminators require an accessory factor called

rho for termination.

Termination The dissociation of the transcription complex and the

ending of RNA synthesis

Occurs at a specific DNA sequence known as the terminator

These sequences often contain self complementary regions

hairpin

Termination These cause the polymerase to pause and

subsequently cease transcription.

Some terminator sequences can terminate transcription without the requirement for accessory factors, whereas other terminator sequences require the rho protein (ρ) as an accessory factor

Transcription in prokaryotesEscherichia coli RNA polymeraseRNA polymerase is responsible for RNA synthesis

(transcription).

The core enzyme, consisting of 2δ, 1β, 1β֨ and 1ω (omega) subunits, is responsible for transcription elongation.

The sigma factor (σ), is also required for correct transcription initiation.

Transcription in prokaryotesThe complete enzyme, consisting of the core enzyme

plus the factor, is called the holo-enzyme SubunitTwo alpha (δ) subunits are present in the RNA

polymerase.

They may be involved in promoter binding.

Transcription in prokaryotes

SubunitOne beta (β) subunit is present in the RNA

polymerase. The antibiotic rifampicin and th streptolydigins bind

to the subunit.

The subunit may be involved in both transcription initiation and elongation.

SubunitOne beta prime (β֨) subunit is present in the RNA

polymerase. It may be involved in template DNA binding.

Heparin binds to the subunit.

Transcription in prokaryotesSigma factor Sigma (σ) factor is a separate component from the core

enzyme.

Escherichia coli encodes several factors, the most common being σ 70.

A σ factor is required for initiation at the correct promoter site.

Transcription in prokaryotes It does this by decreasing binding of the core enzyme

to nonspecific DNA sequences and increasing specific promoter binding.

The factor is released from the core enzyme when the transcript reaches 8–9 nt in length.

Transcription in eukaryotes

Three eukaryotic polymerases transcribe different sets of genes.

Eukaryotic RNA polymerasesRNA polymerase I is located in the nucleoli.

It is responsible for the synthesis of the precursors of most rRNAs.

RNA polymerase II is located in the nucleoplasm and is responsible for the synthesis of mRNA precursors and some small nuclear RNAs.

Eukaryotic RNA polymerasesRNA polymerase III is located in the nucleoplasm. It is responsible for the synthesis of the precursors of

5S rRNA, tRNAs and other small nuclear and cytosolic RNAs.

RNA polymerase subunitsEach RNA polymerase has 12 or more different

subunits.

The largest two subunits are similar to each other and to the subunits of E. coli RNA polymerase.

Other subunits in each enzyme have homology to the subunit of the E. coli enzyme.

Eukaryotic RNA polymerase activities

Like prokaryotic RNA polymerases, the eukaryotic enzymes do not require a primer and synthesize RNA in a 5 to 3 direction.

Unlike bacterial polymerases, they require accessory factors for DNA binding.

The CTD of RNA Pol II

The largest subunit of RNA polymerase II has a seven amino acid repeat at the C terminus called the carboxy-terminal domain (CTD).

This sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser, is repeated 52 times in the mouse RNA polymerase II (phosphorylation).

RNA polymerase IIRNA polymerase II (RNA Pol II) is located in the

nucleoplasm.

It is responsible for the transcription of all protein-coding genes, some small nuclear RNA genes and sequences encoding micro RNAs and short interfering RNAs.

Post transcription modificationsThe pre mRNAs must be processed after synthesis by

cap formation at the 5-end of the RNA and poly(A) addition at the 3-end, as well as removal of introns by splicing

Capping Splicing Poly A tail

PromotersMany eukaryotic promoters contain a sequence called

the TATA box around 25–35 bp upstream from the start site of transcription

It has the 7 bp consensus sequence 5-TATA(A/T)A(A/T)-3

Protein which binds to the TATA box (TBP)

The TATA box acts in a similar way to an E. coli promoter –10 sequence to position the RNA Pol II for correct transcription initiation

The low activity of basal promoters is greatly increased by the presence of other regulatory elements located upstream of the promoter.

SP1 box play an important role in ensuring efficient transcription from the promoter.

Enhancers

• Transcription from many eukaryotic promoters can be stimulated by control elements that are located many thousands of base pairs away from the transcription start site.

• This was first observed in the genome of the DNA virus SV40.

• Enhancer sequences are characteristically 100–200 bp long and contain multiple sequence elements which contribute to the total activity of the enhancer.

1. TFIID bind to the TATA box (TBP associatedfactors or TAFIIs.)2. TBP binds to the minor groove of the DNA at the

TATA box, unwinding the DNA and introducing a 45° bend.

3. TFIID binding to the TATA box is enhanced by TFIIA

4. TFIIB binds to TFIID and acts as a bridge factor for RNA polymerase binding.

5. The RNA polymerase binds to the complex associated with TFIIF.

6. After RNA polymerase binding; TFIIE, TFIIH and TFIIJ associate with the transcription complex in a defined binding sequence

TFIIH phosphorylates the carboxy-terminal domain (CTD) of RNA Pol II.

This results in formation of a processive polymerase

complex.

Thank you