In this article we will discuss about:- 1. Meaning of Transcription in Prokaryotes 2. Mechanism of Transcription in Prokaryotes 3. Reverse Transcription 4. Prokaryotic vs. Eukaryotic Transcription 5. Detection.
Meaning of Transcription in Prokaryotes:
Transcription is the process through which a DNA sequence is enzymaticaly copied by an RNA polymerase to produce a complementary RNA. The synthesis of RNA from a single strand of a DNA molecule in the presence of enzyme RNA polymerase is called transcription.
In other words, it is the process of formation of a messenger RNA molecule using a DNA molecule. Much of the pioneering work on transcription was carried out in prokaryotes, most notably in the bacterium E. coli. These studies laid the foundation for work that was later carried out in the more complex eukaryotes.
The RNA synthesis by RNA polymerase was established in vitro by several laboratories by 1965; however, the RNA synthesized by these enzymes had properties that suggested the existence of an additional factor needed to terminate transcription correctly.
In 1972, Walter Fiers became the first person to actually prove the existence of the terminating enzyme. In 2006, Roger D. Kornberg won the Nobel Prize in Chemistry “for his studies of the molecular basis of eukaryotic transcription.”
The main points related to transcription are listed below:
1. Synthesis:
RNA is synthesized from a DNA template. The RNA is processed into messenger RNA [mRNA], which is then used for synthesis of a protein. The RNA thus synthesized is called messenger RNA (mRNA), because it carries a genetic message from the DNA to the protein-synthesizing machinery of the cell.
The main difference between RNA and DNA sequence is the presence of U, or uracil in RNA instead of the T, or thymine of DNA.
2. Template Used:
The RNA is synthesized from a single template [strand] of a DNA molecule. The stretch of DNA that is transcribed into an RNA molecule is called a transcription unit. A transcription unit codes the sequence that is translated into protein. It also directs and regulates protein synthesis.
The DNA strand which is used in RNA synthesis is called template strand; because it provides the template for ordering the sequence of nucleotides in an RNA transcript. The DNA strand which does not take part in DNA synthesis is called coding strand, because, its nucleotide sequence is the same as that of the newly created RNA transcript.
3. Enzyme Involved:
The enzyme that carries out transcription is called RNA polymerase, and it consists of four kinds of polypeptides, designated α, β, β’ and σ, which are bound together into a complex called a holoenzyme.
4. Genetic Information Copied:
In this process, the genetic information coded in DNA is copied into a molecule of RNA. The genetic information is transcribed or copied, from DNA to RNA.
5. First Step:
The expression of a gene consists of two major steps, viz., transcription and translation. Thus transcription is the first step in the process of gene regulation or protein synthesis.
6. Direction of Synthesis:
As in DNA replication, RNA is synthesized in the 5′ —> 3′ direction. The DNA template strand is read 3′ —> 5′ by RNA polymerase and the new RNA strand is synthesized in the 5’—> 3′ direction. RNA polymerase binds to the 3′ end of a gene (promoter) on the DNA template strand and travels toward the 5′ end.
Mechanism of Transcription in Prokaryotes:
The mechanism of transcription consists of three major phases or stages viz:
(1) Initiation,
(2) Elongation and
(3) Termination.
These are briefly discussed as follows:
1. Initiation:
In bacteria, transcription begins with the binding of RNA polymerase to the promoter in DNA. The RNA polymerase is a core enzyme consisting of five subunits: 2 α subunits, 1 β subunit, 1 β’ subunit, and 1 σ subunit. At the start of initiation, the core enzyme is associated with a sigma factor (number 70) that aids in finding the appropriate -35 and – 10 base pairs downstream of promoter sequences.
The initiation consists of the following steps:
(i) RNA polymerase (RNAP) binds to one of several specificity factors, to form a holoenzyme. In this form, it can recognize and bind to’ specific promoter regions in the DNA. At this stage, the DNA is double-stranded (“closed”). This holoenzyme/wound-DNA structure is referred to as the closed complex.
(ii) The DNA is unwound and becomes single-stranded (“open”) in the vicinity of the initiation site (defined as + 1). This holoenzyme/unwound-DNA structure is called the open complex.
(iii) The RNA polymerase transcribes the DNA, but produces about 10 abortive (short, nonproductive) transcripts which are unable to leave the RNA polymerase because the exit channel is blocked by the cr-factor.
(iv) The a-factor eventually dissociates from the holoenzyme, and elongation proceeds. Most transcripts originate using adenosine-5′-triphosphate (ATP) and, to a lesser extent, guanosine-5′-triphosphate (GTP) (purine nucleoside triphosphates) at the +1 site. Uridine-5′- triphosphate (UTP) and cytidine-5′-triphosphate (CTP) (pyrimidine nucleoside triphosphates) are dis-favoured at the initiation site.
2. Elongation:
In transcription only one strand of DNA [called template strand or non-coding strand] takes part as a template. As transcription proceeds, RNA polymerase traverses the template strand and uses base pairing complementarity with the DNA template to create an RNA copy.
Although RNA polymerase traverses the template strand from 3′ —> 5′, the coding (non-template) strand is usually used as the reference point, so transcription is said to go from 5′ -> 3′.
This produces an RNA molecule from 5′ 3′, an exact copy of the coding strand (except that thymines are replaced with uracils, and the nucleotides are composed of a ribose (5-carbon) sugar where DNA has deoxyribose (one less oxygen atom) in its sugar-phosphate backbone).
In the prokaryotes, the elongation starts with the “abortive initiation cycle”. During this cycle RNA Polymerase will synthesize mRNA fragments 2-12 nucleotides long. This continues to occur until the σ factor rearranges, which results in the transcription elongation complex (which gives a 35 bp moving footprint). The a factor is released before 80 nucleotides of mRNA are synthesized.
3. Termination:
In prokaryotes, two different modes of transcription termination, viz:
(i) Rho-independent and
(ii) Rho-dependent are well known.
These are briefly discussed as follows:
(i) Rho-independent termination:
It is also known as intrinsic transcription termination. It 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 the DNA template.
In the Rho-independent transcription termination, RNA transcription stops when the newly synthesized RNA molecule forms a G-C rich hairpin loop, followed by a run of U’s, which makes it detached the DNA template.
(ii) Rho-dependent termination:
In the “Rho-dependent” type of termination, a protein factor called “Rho” [P factor] is used to stop RNA synthesis at specific sites. This protein binds at a Rho utilisation site on the nascent RNA strand and runs along the mRNA towards the RNA polymerase.
When p-factor reaches the RNAP, it causes RNAP to dissociate from the DNA, terminating transcription. In other words, it destabilizes the interaction between the template and the mRNA, thus releasing the newly synthesized mRNA from the elongation complex.
Reverse Transcription in Prokaryotes:
Synthesis of DNA from RNA molecule in the presence of enzyme reverse transcriptase is referred to as reverse transcription. Reverse transcription was first reported by Temin and Baltimore in 1970 for which they were awarded Nobel Prize in 1975.
Reverse transcription is also known as Teminism. Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA. HIV has an RNA genome that is duplicated into DNA.
The resulting DNA can be merged with the DNA genome of the host cell. The main enzyme responsible for synthesis of DNA from an RNA template 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 synthesizes 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).
Prokaryotic vs. Eukaryotic Transcription:
The process of transcription is the same in both eukaryotes and prokaryotes in several aspects. However, there are some differences in transcription of these two groups as highlighted below.
1. The process is much more complicated in eukaryotes than prokaryotes.
2. In eukaryotes, transcription and translation take place separately in nucleus and cytoplasm respectively while in prokaryotes both processes take place simultaneously in the cytoplasm.
3. The eukaryotic mRNA contains introns and hence needs modification before taking part in protein synthesis. In prokaryote, the mRNA does not require modification.
4. Eukaryotes have DNA in the nucleus, whereas in prokaryotes DNA is in the cytoplasm.
Detection of Transcription in Prokaryotes:
Transcription can be measured and detected in a variety of ways.
The commonly used methods of detecting transcription are given below:
1. Nuclear Run-on assay, measures the relative abundance of newly formed transcripts.
2. RNAse protection assay and ChlP-Chip of RNAP, detect active transcription sites.
3. RT-PCR, measures the absolute abundance of total or nuclear RNA levels, which may however-differ from transcription rates.
4. DNA microarrays measures the relative abundance of the global total or nuclear RNA levels, which may however differ from transcription rates.
5. In situ hybridization, detects the presence of a transcript.