TRANSCRIPTION IN PROKARYOTES (steps of transcription part 2)

There are 3 steps of Transcription: a) Initiation b) Elongation c)Termination.

INITIATION

Initiation is further divided into three phases : 

1. Isomerization   2. Abortive Transcription  3. Promoter Escaping

ISOMERIZATION:

Transition from closed to open complex involves structural changes in enzyme. This reveals the template and non-template strands. This melting occurs between -11 to +2 position. And does not require ATP hydrolysis instead result of conformational change.

Two bases in non-template strand of -10 element (A11 and T7) flip out from stacking and insert into pockets within the sigma protein. By stabilizing the single stranded form of -10 element, these interactions drive melting of promoter region. 

THERE ARE FIVE CHANNELS INTO THE ENZYME:

1. NTP-uptake channel: It allows ribonucleotides to enter the active center to incorporate into RNA chain.
2. RNA exit channel: It allow growing RNA chain to leave the enzyme.
3. Downstream DNA channel: Downstream ds DNA enter the active center cleft through this.
4. Non-template strand (NT) channel: Non-template strand exit the active and travel across surface of enzyme.
5. Template strand channel:  Template strand follow a path through the active cleft and exits through T channel.

The double helix reforms at -11 in upstream DNA behind the enzyme.

STRUCTURAL CHANGES:

• Pincers at front of enzyme clamp down tightly on downstream DNA.
• Major shift in position of alpha-NTD (amino terminal region of sigma).

When not bound to DNA, sigma region 1.1 lies within the active site blocking the path that in open complex, followed by template strand. In open complex, 1.1 shifts some 50 A and found on outside of enzyme.

Region 1.1 of sigma is highly negatively charged. Thus, it act as molecular mimic of DNA. The space in active site is highly positively charged.

Why most transcripts start with the same nucleotide?

For the requirement of specific interactions between enzyme and initiating nucleotide. The structure of open complex shows that sigma region 3/4 linker interacts with the template strand, organizing it in correct conformation and location to allow initiation.

During initial transcription, RNA Pol. remains stationary and pulls downstream DNA into itself. This process is called 'scrunching process'.

The active center of RNA Pol. is translocated forward relative to the DNA template and synthesize short transcripts before aborting, then repeats this cycle until it escapes the promoter. Three models for figuring this were as follows:

• Transient excursions : Polymerase moves along the DNA.
• Inch worming: Front part of enzyme moves along the DNA, but back part remain at promoter.
• Scrunching : Enzyme remains stationary and pulls the DNA into itself.

ABORTIVE TRANSCRIPTION:

Initially primary transcript of 7-8 nucleotides is formed which is not able to push the linker sigma 3/4 region present at RNA exit channel and release from the active site. This process is repeated until more than 8 nucleotides are formed.

Once 9-10 length is formed, the transcript cannot accommodate within the region where it hybridizes to DNA and must start threading into RNA exit channel.

Promoter escape is associated with breaking of all interaction between Pol. and promoter and any regulatory proteins.

Scrunching is a way to store and mobilize energy during transcription initiation.

PROMOTER ESCAPING:

During promoter escaping, release of sigma 3/4 region weak the elongating complex (RNA Pol. + transcript). Since sigma is responsible for association of RNA Pol. to promoter region. Once the sigma 3/4 region is released, it weakens the association between RNA Pol. and promoter.

ELONGATION:

• 8-9 nucleotides remain bound to the DNA template.
• DNA passes through enzyme and enter the catalytic site where the strand separate and ribonucleotides enter the active site to their channels and adding to the growing channel.
• The enzyme add one nucleotide at a time to the growing RNA transcript.
• The enzyme steps forward as a molecular motor.
• The size of the bubble i.e., length of DNA is not double helical, remains constant as 1 base pair separated ahead and 1 base pair is formed.

PROOFREADING:

RNA Pol. performs two types of proof reading:

1. PYROPHOSPHOROLYTIC EDITING:

• Enzyme uses its active site, in a simple back reaction, to catalyze the removal of an incorrectly inserted ribonucleotide by reincorporation of PPi.
• The enzyme then incorporates another ribonucleotide in its place in growing RNA chain.

2. HYDROLYTIC EDITING:

• The Pol. backtracks by one or more nucleotides and cleaves the error-RNA.
• It is stimulated by Gre factors, which enhances hydrolytic editing function and serves as elongation stimulating factors means (Pol. elongates efficiently and overcome 'arrest'.)

RNA Pol. can become arrested and need removing:

In case of damaged DNA strand, the consequences of arrest cause roadblock to other Pol attempting to transcribe same gene. Transcription-coupled repair (TRCF) proteins has an ATPase activity. It binds dsDNA upstream of Pol. and use ATPase motor to translocate along DNA until it encounters RNA Pol. The collision pushes Pol. forward, allowing it to restart elongation or dissociation of ternary complex of RNA Pol. , template DNA and RNA transcript. This terminates transcription.

TERMINATION:

It is a normal and important function at the end of genes. Sequences called terminators trigger the elongating polymerase to dissociate from DNA and release RNA chain it has made. It is of two types:

Rho-dependent Termination:

• It requires rho proteins and contains rut sites.
• Rho is ring shaped protein with 6 identical subunits, binds to ssRNA as it exits the polymerase.
• This protein has ATPase activity. Once attached to the transcripts, Rho uses ATP hydrolysis energy to induce termination.
• Rho binds to RNA Pol. throughout the transcription cycle.
• The role of translocation by Rho is to tighten the resulting RNA loop and when sufficiently tight, Pol. elongation ceases.
• The rut sites for rho utilization consist of stretches of 40 nucleotides that do not fold into secondary structure. They remain single stranded and are rich in C residues.
• Rho only terminates those transcripts still being transcribed beyond the end of gene or operon.
• Rho fails to bind any transcript that is being translated.

Rho-independent Termination:

• There is no involvement of other factors.
• Rho-independent terminators are also called intrinsic terminators.
• It consist of two sequence elements: a short inverted repeats of 20 nucleotides , followed by stretch of eight AT base pair.
• These elements affect the Pol. after transcription..
• After transcription, the resulting RNA can form a stem loop structure (hairpin) by base-pairing itself. This hairpin causes termination by disrupting the elongation complex.
• The hairpin works efficient terminator when followed by stretch of AU base pair.
• Because AU base pair are weakest of all base pair they are more easily disrupted by the effect of stem-loop on transcribing Pol. 
• And thus RNA will more readily dissociate.