PROKARYOTIC TRANSCRIPTION

Submitted By: Priyanka Bissa M.Sc (prev) 2007-08

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Central dogma

DNA RNA PROTEIN

INTRODUCTION

• Transcription is the process through which a DNA sequence is enzymatically copied by an RNA polymerase to produce a complementary RNA. So to say, it is the transfer of genetic information from DNA into RNA.

HISTORY

• The first suggestion that RNA is derived from the transcription of DNA was made in the 1950s by Eliot Volkin and his colleagues at the Oak Ridge national Laboratory.

Prokaryotes Transcription

Transcription divided into three stage:- - Initiation - Elongation - Termination

Prokaryotic transcription occurs in the cytoplasm

Prokaryotic Transcription

The following steps occur, in order, for transcription

Initiation:-• The following steps occur, in

order, for transcription initiation:• RNA polymerase (RNAP)

recognizes and specifically binds to the promoter region on DNA. At this stage, the DNA is double-stranded ("closed"). This wound-DNA structure is referred to as the closed complex.

• The DNA is unwound and becomes single-stranded ("open") in the vicinity of the initiation site (defined as +1). This unwound-DNA structure is called the open

complex.

• The RNA polymerase transcribes the DNA, but produces about 10 abortive transcripts which are unable to leave the RNA polymerase because the exit channel is blocked by the σ-factor.

• The σ-factor eventually dissociates from the holoenzyme, and elongation proceeds.

Elongation

• The σ-factor eventually dissociates from the holoenzyme, and elongation proceeds.

• Transcription can involve multiple RNA polymerases, so many mRNA molecules can be produced from a single copy of the gene.

• This step also involves a proof reading mechanism that can replace an incorrectly added RNA molecule.

Termination

The end of RNA synthesis is known as termination. Termination occur when RNA-DNA hybrid region is forced to separate, thereby releasing the newly made RNA transcript as well as the RNAP. . Termination mechanisms are well known:• The DNA template. Intrinsic termination (also called

Rho-independent transcription termination) involves terminator sequences within the RNA that signal the RNA polymerase to stop. The terminator sequence is usually a palindromic sequence that forms a stem-loop hairpin structure that leads to the dissociation of the RNAP from

Stem-loop

• It occurs when two regions of the same molecule, usually palindromic in nucleotide sequence, base-pair to form a double helix that ends in an unpaired loop. The resulting lollipop-shaped structure is a key building block of many RNA secondary structures

• The hairpin loop forms in an mRNA strand during transcription and causes the RNA polymerase to become dissociated from the DNA template strand.

• Rho-dependent termination uses a termination factor called ρ factor(rho factor) which is a protein to stop RNA synthesis at specific sites. This protein binds at a rho recognition site on the nascent RNA strand and runs along the mRNA towards the RNAP.

Prokaryotic vs. eukaryotic transcription

• Prokaryotic transcription occurs in the cytoplasm.

• Eukaryotic transcription is primarily localized to the nucleus, where it is separated from the cytoplasm by the nuclear membrane

Transcription factories

• Transcription factories can also be localized using fluorescence in situ hybridization, or marked by antibodies directed against polymerases.

• There are ~10,000 factories in the nucleoplasm of a HeLa cell, among which are ~8,000 polymerase II factories and ~2,000 polymerase III factories.

• Active transcription units are clustered in the nucleus, in discrete sites called ‘transcription factories’.

Reverse transcription

Scheme of reverse transcription• Some viruses (such as HIV, the cause of AIDS), have the ability to

transcribe RNA into DNA in order to see a cell's genome. • The main enzyme responsible for this type of transcription is called

reverse transcriptase. In the case of HIV, reverse transcriptase is responsible for synthesizing a complementary DNA strand (cDNA) to the viral RNA genome.

• An associated enzyme, ribonuclease H, digests the RNA strand and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure.

• This cDNA is integrated into the host cell's genome via another enzyme (integrase) causing the host cell to generate viral proteins which reassemble into new viral particles. Subsequently, the host cell undergoes programmed cell death (apoptosis).