Transcription

Transcription

Vipin Shankar

The Central Dogma

The information on the DNA has to be converted to the protein quotient of the cell.

This is carried in a two step process.

DNA is first transcribed to RNA. The RNA is then translated to

proteins.

Transcription

Chemically and enzymatically similar to DNA replication.

Both involve the synthesis of a new strand of nucleotide complimentary to a DNA template strand.

The most notable difference between the 2 is that, in transcription, the new strand is a RNA.

Transcription v/s Replication

RNA Polymerase: the enzyme that catalyze the reaction, does not require a primer.

The RNA product does not remain base paired to the template DNA.

Transcription is less accurate than replication. (error rate of 1 in 10,000 compared to 1 in 10,000,000)

Transcription v/s Replication…

Transcription selectively copies only certain parts of the genome and makes anything from one to several hundred (or even thousand) copies of any given section.

RNA Polymerases

Essentially performs the same reaction in all cells from bacteria to humans.

All RNA Polymerases, share some common features.

The cellular RNA Pol is a multi-subunit complex protein.

Bacteria has a single RNA Pol, Eukaryotic cells have 3; RNA Pol I, II & III.

RNA Polymerases…

RNA Pol-II carries out transcription of mRNAs in eukaryotes.

RNA Pol-II & III are involved in transcribing specialized RNA –encoding genes.

RNA Polymerases…

The bacterial core enzyme is composed of 2 copies of subunits, one each of , ’ & subunits.

The shape of each enzyme complex resembles a crab claw.

The two pincers of the crab claw are made up of the two large subunits & ’.

RNA Polymerases…

The active site is at the base of the two pincers called the active cleft.

The active cleft binds 2 Mg2+ ions. There are various channels that

allow DNA, RNA and ribonuclotides into and out of the enzymes’ active cleft.

Steps in transcription

3 phases are identified. Initiation Elongation Termination

Initiation

A region called the Promoter on the DNA binds to RNA Pol.

The promoter-polymerase complex undergoes structural changes.

The DNA around the point where transcription starts unwinds producing a Transcription Bubble.

Initiation…

Transcription always occurs in 5’ to 3’ direction.

Only one of the DNA strand act as a template.

The RNA Pol binds to the promoter in a defined orientation, and the same strand is transcribed.

The choice of the promoter determines which strand is to be transcribed and also the rate of transcription.

Initiation…

Involves 3 steps. Initial binding of RNA Pol to a promoter to

form a closed complex. The closed complex undergoes transition

to the open complex in which the DNA strand separates for over 14bp.

The unwinding of the double helix, initiates transcription.

The first two rNTPs are brought to the active cleft and the system begins polymerization.

Initiation…

The enzyme then moves along the template Opening the double helix ahead of the site of

polymerization. Incorporating the complimentary rNTP. Re-annealing the double helix behind the

polymerization site. The incorporation of the first 10 rNTPs is

inefficient. Once an enzyme gets further than 10bp, it

is said to have escaped the promoter. At this point it has formed the Stable

Ternary Complex.

Elongation

Once the RNA Pol has synthesized a short segment of RNA, it enters the elongation phase.

This requires a conformational change in RNA Pol.

During elongation the enzyme performs Unwinds DNA in front & re-anneals it

behind. Dissociates the RNA chain from the

template as it moves along. Performs proof reading function.

Termination

Once the polymerase has transcribed the length of the gene it stops and releases the RNA product.

In some cells there are well characterized termination sequences that trigger termination.

Transcription in Bacteria

An additional protein called the -factor.

The form of RNA Pol with the -factor is called the holoenzyme.

In the case of E. coli, the predominant -factor is called 70.

70 recognizes the promoter.

Transcription in bacteria…

The promoter region is a stretch of 2 conserved sequences at the -10 and -35 region.

The consensus sequence can be derived by comparing the promoter regions of various organisms.

Promoters with sequences closer to the consensus sequence are strong promoters.

Transcription in bacteria…

An additional element that binds to RNA Pol is found is some strong promoters – UP-element.

-factor mediates RNA Pol binding on the promoter

The 70 has a helix-turn-helix motif.

One of the helices interacts with the -35 region.

The -10 region is recognized by another helix.

DNA melting is initiated within the -10 region.

-factor mediates RNA Pol binding on the promoter…

The UP-element is not recognized by 70.

This is recognized by the CTD (c terminal of subunit).

Transition to the open complex

The transition involves Unwinding of the double helix

between -11 and +3 regions. Conformational change in the

protein. In case of the 70 bearing

holoenzyme, this is called isomerization and does not require energy.

Transition to the open complex…

This happens due to spontaneous conformational change.

Isomerization is irreversible. Completion of isomerization

guarantees initiation of transcription.

RNA Pol does not require a primer

The initiating rNTP is brought into the active cleft and is held stably on the template.

The next NTP is then presented with the correct geometry and polymerization is carried out.

The enzyme makes specific interactions with the initiating rNTP.

Under most conditions a rATP is used as the initiating rNTP.

Abortive initiation

Once RNA synthesis begins, RNA Pol initially synthesizes short RNA (<10nt in length).

Instead of being elongated these are released and RNA Pol restarts with a new RNA.

This is probably because of the positioning of the -factor, which hinders the RNA exit channel.

The -factor later dissociates, and elongation begins.

The elongating polymerase is a processive machine During elongation

The double helix DNA enters the enzyme between the pincers.

At the opening of the catalytic cleft the double helix is unwound, the strands separate and follow different paths and are later rejoined behind the elongating polymerase.

The rNTPs enter through a channel and are added to the growing RNA.

Only 8 or 9 NTPs of the growing RNA remain base paired to the template.

The remainder of the RNA is pealed off and are directed out through the RNA exit channel.

..nnnn.

Proofreading

2 methods. Pyrophosphorolytic editing:

A simple back reaction. Removes an incorrectly inserted rNTP

and reincorporates the correct one. Hydrolytic editing:

Backtracks one or more NTPs and cleaves the RNA product, removing the error containing sequence.

Stimulated by Gre factors.

Termination in bacteria

Sequences called terminators trigger termination

2 different modes Rho-independent Rho-dependent

Rho-independent termination

Also called intrinsic termination. The terminator sequence

comprises of an inverted repeat. This is followed by a a stretch of

about 8 A:T base pairs. These elements do not affect RNA

Pol until after they are transcribed. These elements function in the

RNA rather than in the DNA

Rho-independent termination…

When RNA Pol transcribes an inverted repeat, the resulting RNA forms a hairpin structure.

The hairpin causes termination by disrupting the elongation complex.

Rho-dependent termination Less well characterized terminator

sequences. Requires the action of -protein. This is a ring shaped protein with 6

identical subunits. Binds to the single stranded RNA at

sites called rut (Rho utilization sites). -protein has ATPase activity. Once attached to the transcript, it uses

the energy derived from ATP to wrest the RNA from the polymerase complex.

Transcription in eukaryotes

Eukaryotes have 3 different polymerases.

Several initiation factors are required General transcription Factors (GTFs).

Additional factors Mediator complex DNA binding regulatory proteins Chromatin modifying enzymesAre also required.

Types of RNA polymerases and their function

Types of Transcription factors

Promoters

The core promoter refers to the minimal sequence elements required for accurate transcription initiation by Pol II.

Typically 40 nts long, extending either upstream or downstream.

4 elements found in ek promoters are TFIIB recognition element. The TATA Box. The Initiator (Inr). The downstream promoter element (DPE).

Regulatory elements and factors recognizing them

RNA Pol II forms a pre-initiation complex TFIID binds to the TATA box via the TBP. This association distorts the -10 region and

slightly unwinds the DNA double helix. The TBP-DNA complex recruits TFIIA, TFIIB,

TFIIF together with polymerase. The polymerase is in complex with TFIIE,

TFIIH and the mediator complexes which bind upstream to the TATA box.

This assembly of proteins on the DNA double helix is called the pre-initiation complex.

The pre-initiation complex… The DNA melting is mediated by TFIIH. Action of TFIIH is similar to Helicase

and requires energy. Once the pre-initiation complex is

formed the polymerase begins to add rNTPs.

The system first enters an abortive initiation phase similar to that in prokaryotes.

The phosphorylation of RNA Pol-II CTD results in escape from the promoter and the triggering of elongation.

Elongation Elongation requires specific factors

called elongation factors. These factors are of 2 types

Those that stimulate elongation. Those that bring about RNA editing.

Both these factors are attached to the RNA Pol-II CTD.

There is an overlap of elongation and RNA editing.

RNA editing is simultaneous with elongation.

Termination

Termination is less understood in eukaryotes.

It is proposed that the 3 polymerases use different mechanisms for termination.

Pol-I uses a mechanism similar to Rho-dependent termination.

Pol-III terminates after transcribing a termination sequence which contains a string of Us.

..nnnn n nn n .