TRANSCRIPTION IN PROKARYOTES

SUBMITTED BY:
Aditya Kanwal- 2012BT09 M.Tech Biotechnology MNNIT

INTRODUCTION:
Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA by the enzyme RNA polymerase. Transcription can be shown as(for example in trp operon):

In transcription, the template strand (denoted 3' ---> 5') is utilized to synthesize RNA molecules. Transcription and translation are coupled in Prokaryotes and occur simultaneously as there is no nucleus. Polycistronic RNA is synthesized i.e. multiples genes are transcribed in one chain. tRNA and rRNA primary transcripts go through a maturation phase in which some part of their 3' ends is cleaved off. They do not code for proteins but only assist in the process. mRNAs comparitively have lesser half life times of about 1- 3 minutes. They are the ones that code for proteins.

RNA Polymerase:
It is the enzyme that catalyses polymerisation of RNA from a DNA template. Affinity between RNA polymerase and DNA duplex is created by electrostatic ineractions between basic protein chain and acidic DNA. Only one type of RNA Polymerase is found in prokaryotes and in E. coli it is of 460 Kda. It has 5 subunits:
CODING GENE rpo A rpo B rpo C rpo Z rpo D FUNCTION Enzyme assembly and promoter recognition. Catalytic core Catalytic core Enzyme assembly Recognition of Promoter.

SUBUNITS 2 - subunits - subunit ' - subunit - subunit - subunit

BACTERIAL SIGMA FACTORS:

- Sigma factors are transcription factors - Different sigma factors bind RNA Polymerase and recognize specific -10 ,-35 sequences - Helps melt DNA to expose transcriptional start site E. coli has 7 sigma factors as shown below
Sigma subunit RpoD RpoN RpoS RpoH RpoF RpoE FecI Type of gene controlled Growth/housekeeping N2; stress response Stationary phase, virulence Heat shock Flagella-chemotaxis ? Ferric citrate transport # of genes controlled ~1000 ~15 ~100 ~40 ~40 ~5 ~5

THE PROMOTER:
Promoters are those regions of DNA that create sites for the binding of RNA polymerase. They contain some consensus sequences: -10 region: 'TATAAT' -35 region: 'TTGACA'

-10 region is the site of the melting of DNA helix. -35 region is the site that provides binding energy to RNA polymerase.

STEPS OF TRANSCRIPTION:
The process of Transcription can be divided into three steps: 1) Initiation 2) Elongation 3) Termination

OVERVIEW:

INITIATION:
It can further be divided into three phases: 1) Closed complex formation: In this, RNA polymerase binds to the promoter with the help of factor. The binding is such that DNA remains double stranded and Polymearse is bound only at one face of the helix.

2) Open complex: After the melting of DNA at -10 site of the promoter, a transcription bubble is formed and RNA polymerase grips the template strand. The process is called Isomerization. 3) Stable Ternary complex: Ribonucleotides are then incorprated into chains however, a phase of abortive initiation occurs in which short transcripts of about 10 nucleotides are synthesized and subsequently released due to the blockage created by factor in the RNA exit channel of RNA polymerase. When this factor is displaced, only then the RNA chain is synthesized in a proper way and a stable ternary complex is formed.

ELONGATION:
Two things happen in the elongation phase: 1) Growth of RNA chain: After a stable ternary complex is formed, RNA synthesis is continued. The addition of ribonucleotides is catalyzed by the and ' subunits of RNA polymerase core enzyme. If somehow the polymerization gets stalled, it can be restarted with the help of Gre factors A and B. Also, to facilitate polymerization, DNA Gyrases introduce negative supercoils in front of the RNA polymerase and Topoisomerase I removes negative supercoils behind the RNA polymerase.

2) Proofreading: i) Phosphorolytic Editing: In this, the enzyme uses its active site, in a simple back reaction, to remove the wrong nucleotide by reincorporation of inorganic phosphates (PPi). ii)Hydrolytic Editing: In this, Gre factors are utilized to backtrack one or more nucleotides and removing the error containing sequence.

TERMINATION:
Sequences called Terminators trigger the elongating polymerase to dissociate from DNA and release the RNA chain it has made. Termination in Prokaryotes can be of two types: 1) Intrinsic or Rho independent terminators: In this case, the DNA codon consists of two sequence elements: * A short inverted repeat of 20 nucleotides. * An 8 bp long stretch of A:T sequence. When polymerase transcribes this inverted repeat sequence, the resulting RNA forms a hairpin like structure.

Further, the A:T rich sequence is transcribed resulting in A:U rich sequence on RNA which form the weakest interactions and thus the RNA gets detached also releasing the polymerase.

2) Extrinsic or Rho Dependent terminators: Rho proteins are hexameric ring shaped proteins, each monomer having two domains. It binds to RNA as it exits at Rho utilization sites (rut) and moves to encounter RNA polymerase. A stem loop structure upstream of the terminator region pauses the RNA Polymerase, when Rho factor

REFERENCES:
- Watson J.D. , Molecular Biology Of The Gene ,Pearson Education India, (2004) - Lewin B. , Genes X. Pearson Prentice Hall(2009) - Cooper G.M. ,The Cell: A Molecular Approach. 2nd edition. (2000) - http://en.wikipedia.org/wiki/Prokaryotic_transcription - http://www.msf.bio.ic.ac.uk/sigma.php - http://en.wikipedia.org/wiki/Transcription_genetics