Friday, 21 May 2021

Transcription process in Prokaryotes

  The transfer of genetic information from double standard template DNA molecule to a single standard RNA molecule by usual process of complementation base pairing catalysed by enzyme RNA polymerase.
  Transcription occurs unidirrectionally in which RNA (transcript) is synthesized from the 5' to 3' direction.

Introduction

Crick in 1958 reported, replication is auto catalytic function of DNA. Transcription and translation are heterocatalytic function of DNA. 

• Central doma : This name was given by Crick to the 2 step process.
DNARNAProtein
DNA to RNA by Transcription and RNA to DNA by Translation.

• Transcription Unit
  Transcription is a selective process. Each transcript segment of DNA is called transcription unit there are two types of transcription unit :
a. monocistronic transcription unit
In Eukaryotes, transcription unit typically carries the information of just one gene.
b. Polycistronic transcription unit  
In prokaryotes, a set of adjacent genes is transcribed as a unit termed Polycistronic transcription unit.

• Transcription occurs throughout the cell cycle depends on need.

• Transcription involves rewriting of genetic message in DNA and RNA molecule and results in the formation of mRNA (messenger RNA) complementary to the gene sequence of one of two strands of double stranded DNA helix.

• The lifespan of mRNA is very short, it present only during protein synthesis. 

• Transcription is catalyzed by RNA polymerase in prokaryotes and eukaryotes.

• RNA synthesis takes place in 5'➞3' direction. RNA polymerase can add nucleotides to the 3' OH end of the polynucleotide.
- Start point is the first base pair at which transcription starts. RNA polymerase moves along template and elongate until reaches terminator sequence.

• During transcription only 3' to 5' strand (one strand) of DNA is transcribed. This strand is called coding strand which is comple complementary to the template strand from which RNA is transcribed 5' to 3' strand.
The template strand or non coding strand is the 3' to 5' strand from which the RNA is transcribed.

RNA synthesis can start without primer (De-novo). First nucleotide at 5' end of RNA transcript is purine triphosphate (GTP or ATP).

• Primary transcript :
   The immediate product of transcript is called the primary transcript, that is processed to yield either single type or more than one type of mRNAs depending on transcription unit is simple or complex.

Mechanism of Transcription in Prokaryotes

Requirements
• Template DNA
• Ribonucleotides (GTP,CTP, UTP, ATP)
• Mg+² ions (as cofactor).
• RNA polymerase (DNA dependent RNA polymerase).

RNA polymerase
• This is a DNA dependent RNA polymerase
• Discovered by Samuel B. Weiss and Jerard Hurwitz in 1960.
• In prokaryotes different types of RNA ( mRNA, rRNA, tRNA) are synthesized by single RNA polymerase.

  Bacterial RNA polymerase is a complex holoenzyme (4,80,000 M.W.). It is made up of core enzyme + Sigma(σ) subunit.

RNA Polymerase Subunits and Functions
Subunit Function
 2α Assembly of core enzyme & Promoter recognition
 β Catalytic centre
 β' Catalytic centre
 ω Assembly of RNA polymerase β' unit to be stable
Sigma 
Subunit
Promoter recognition & transcription initiation
Core enzyme : formed of 5 chain (α,α,β,β',ω). It binds non specifically to DNA template strands and catalyse process of chain elongation.
Sigma subunit(σ) is a loosely attached to the core enzyme. It recognises start signal on DNA and directs core enzyme to bind promoter region.

• RNA polymerase lack proofreading 3' to 5' exonuclease activity.
  In every 10⁴ to 10⁵ nucleotide added, a mistake may be made for one nucleotide it is not serious because of high turnover and wobble pairing during transcription.

Operon in prokaryotes

  Prokaryotic DNA is a polycistronic. The length of DNA transcribed into single functional RNA molecule is known as operon in prokaryotes, the segment of operon contains,
- a promoter region
- a short Codon or initiation sequence
- a colony segment
- a terminator sequence

Prokaryotic Pramoter
• Promoter region is a upstream regulatory DNA segment. The initiation codon of gene located before 5' end off coding DNA.
- Strong promoter supports high rate of transcription initiation.
• RNA polymerase binds at Pramoter site.
• Pramoter is highly variable but has 2 to 3 important functional sequence.

Promoter in Prokaryotes

1) -10 Sequence or Pribnow box :
- This is a Consensus sequence of 6 to 10 bases (TATAAT).
- Located upstream to initiation site
- It orients RNA polymerase on promoters
- This sequence helps the enzyme to locate the precise bases at which transcription should initiate. second.

2). -35 region
- A consensus sequence about 35 base above the initiation site (TTGACA)
- Sigma (σ) subunit of RNA polymerase get bound at this site.

3). Upstream promoter element
- AT rich recognition element in promoter
- present only in certain highly expressed genes
- located between -40 to -60 base sequences from initiation codon.

Transcription process in prokaryotes :

Transcription process in Prokaryotes can be divided in 3 step
1. Initiation
2. Elongation and
3. Termination

1). Initiation of transcription :
• Only RNA polymerase (holoenzyme) can initiate transcription.
• Sigma factor recognised consequence sequences (-35 & -10 region of promoter)
• Sigma factor changes the DNA binding properties of RNA polymerase. so that it's affinity for a consensus sequence in promoter is increases.
• The core enzyme does not differ between promoter and other DNA sequence.

Transcription bubble

Closed binary complex :

• RNA polymerase holoenzyme binds at the promoter.
• The enzyme first form a closed complex in which the two DNA strands remain fully base paired.
• RNA polymerase covers 75-80 base pair extending from -55 to +20 bp.

Open reading complex :
• In the next step closed complex is converted to open complex. In this DNA helix open by meeting of short DNA sequence bound by enzyme.
• The template DNA is expressed for initiation of an RNA chain.

• The next step is to add first two nucleotides than a phosphodiester bond forms between them. This generates tertiary complex that contain RNA, DNA and enzyme which is generate transcription bubble.

2). Elongation of transcription
• Sigma factor now dissociate and only core enzyme moves along.
• Elongation involves movement of transcription bubble. As the enzyme moves it unwinds the DNA helix to expose a new segment of template in single stranded DNA.
• Nucleotides are covalently added to 3' end of growing RNA chain forming RNA-DNA hybridize
• The length of RNA-DNA helix with in open complex is 8-9 base pair.
• Overall reaction rate is 30 to 50 nucleotide per second at 37°C.

Direction of Transcription

During its nucleotides edition
- Beta and gamma phosphates are removed from incoming nucleotide.
- OH group is removed from 3' carbon of nucleotide present at the end of the chain.
- During the elongation when RNA polymerase transcribes DNA unwinding and rewinding occurs.
- As RNA polymerase moves ahead generate positive supercoiling ahead (Gyrase removes the positive supercoils and develop negative supercoiling).
- As RNA polymerase moves ahead generate negative supercoiling behind ( Topoisomerase I  removes negative supercoiling).

3). Termination of transcription
• Termination of prokaryotic gene transcription is signalled by controlling elements called terminators.
• At this point, the enzymes stop adding nucleotides to the growing RNA and release the completed product and dissociates from the DNA template.

Terminators are 2 type
1) Intrinsic Terminator
2) rho(ρ) dependent Terminator

1) Intrinsic Terminator
• Intrinsic terminator include palindromic sequences that form hairpin structure by forming a complementary base pairing.

The inverted repeats forming stem and loop structure and terminal U-sequence at the 3 terminal or mRNA molecule

- Hairpins vary in length from 7 to 20 basepairs.
- The stem loop structure includes a G-C rich region and is followed by a U-rich region.
   Typical distance between hairpin and U-rich region is 7-9 bases.

2) Rho(ρ) dependent Terminator
• Rho(ρ) dependent Terminator sequence require activity of a protein called Rho(ρ).
- Rho is an ATP dependent RNA stimulated helicase that disrupt the new ones and RNA DNA complex.
- It binds to RNA at rut site and translocate along RNA in 5' to 3' direction until riches RNA DNA hybrid in RNA polymerase where is release RNA from DNA
- Rut site is rich in C residues and  poor in G residues.

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