Prokaryotes (G: pro-before; karyon- nucleus) are simple, single-cell organisms, which lack a distinct membrane-bound nucleus. These include bacteria and archae (fig 1).
Due to the absence of nucleus, replication, transcription and translation do not have distinct location and all three can occur simultaneously in the cytoplasm.
Like replication in prokaryotes, the transcription too, can be divided into three distinct phases: Initiation, Elongation and termination.
1. Initiation:
Initiation of transcription begins at the promoter, the region where the transcription enzyme RNA polymerase binds the DNA. The promoters are usually located on the upstream of the genes to be transcribed. The promoters of the prokaryotes differ in their sequences, though there are two conserved sequences observed. These two sequences are located approximately 10 and 35 base pairs upstream of the transcription start site.
The transcription start point, which the first base transcribed is termed the +1 site or the initiation site. The region on the upstream of this initiation site is denoted by a negative (-) sign and the ones on the downstream are denoted by a positive (+) sign.
Hence the conserved upstream sites are referred to as -10 and -35 regions. The -10 region has the conserved sequence of TATAAT and the -35 region has TTGACA sequence (fig 2).
The σ subunit of RNA polymerase (RNA pol) binds specifically to both, the -35 and -10 regions, and loads the RNA pol onto the promoter. The binding of the RNA pol to the promoter results in formation of a closed-promoter complex (fig 3).
The RNA pol then interacts with the DNA and unwinds around 15 bases long region at the initiation site to create a transcription bubble, collectively called open-promoter complex. As the initiation site unwinds, single-stranded DNA is exposed and can be used as a template for transcription (fig 3). The strand which is transcribed is known as the template strand or antisense strand. The other strand is called as coding strand or sense strand (fig 1).
RNA pol initially initiates abortive transcription, wherein short cycles of synthesis and release of short RNA transcripts of approximately 10 nucleotides take place till RNA polymerase-promoter initial transcribing complex leave the promoter. The RNA pol escapes the promoter and initiates productive synthesis of RNA, forming RNA polymerase-DNA elongation complex (fig 3).
(Just for info: Read more about Abortive transcription.)
2. Elongation:
RNA polymerase-DNA elongation complex is stable and processive. It carries out transcription on an average rate of 30 – 100 nucleotide/sec.
RNA polymerase is the principle protein, which adds the nucleotides. In most prokaryotes, a single type of RNA pol transcribe all types of RNA. The core RNA pol is made up of 5 subunits, which are conserved in prokaryotes as well as eukaryotes. Prokaryotic core enzyme consists of two copies of α, one copy of each- β, β’, and ω subunits (fig 4). The core enzyme can bind the template DNA and synthesize RNA.
σ factor binds the core enzyme to form the holoenzyme and helps in recognising the promoter.
α subunit interacts with various transcription factors and helps regulate transcription. It also has DNA binding site with help of which it binds upstream promoter DNA. The RNA pol has two α subunits forming a homodimer which binds β and β’ subunits.
β and β’ subunits are the largest subunits which form the catalytic center of RNA synthesis and have binding sites for double-stranded downstream DNA, DNA/RNA hybrid and RNA.
The ω subunit has been linked recently with RNA pol stability and specificity in Staphylococcus aureus.
σ factor associates with the core enzyme for promoter recognition and it dissociates from the core enzyme once RNA pol starts processive RNA synthesis.
(Just for info: Much more on Prokaryotic RNA Polymerase)
RNA pol catalyzes the polymerization of ribonucleoside 5′-triphosphates (NTPs) using the DNA as template. The ribonucleoside triphosphates are added to the 3′ end, i.e. mRNA synthesis takes place in the 5′ to 3′ direction. This extension of RNA chain occurs de novo and doesn’t need a primer.
In prokaryotes, the genomic DNA usually contain no introns and the transcript synthesized may contain information for more than one protein. Such transcripts are called as polycistronic mRNA.
3. Termination:
The transcription has to be terminated to release the newly synthesized mRNA. The termination is brought about by two different mechanism, one is rho independent or RNA-based and the other is rho dependent or protein-based.
• Rho independent or RNA based:
RNA based termination employs intrinsic (in RNA structure itself) terminators having two structural features, GC-rich inverted repeat with several intervening nucleotides, followed by a stretch of U residues in the transcribed RNA.
When the GC-rich inverted repeats are transcribed, the RNA regions with self complementary sequences base-pair with one another and form a stem-loop structure (Fig 5). This stem-loop structure interacts with the RNA polymerase and causes it to pause.
The following stretch of U residues at the 3′ end of the transcript, which are weakly base-paired with the A residues of the template DNA, create a very unstable region.
The stem loop followed by the weakly bond stretch together leads to the release of the RNA transcript from the transcription complex.
(Just for info: Read a paper on Rho independent termination.)
• Rho dependent or protein-based:
This mechanism was first recognized in λ-phage DNA. It involves a protein called Rho factor, which is a hexameric protein (fig 6). Rho is a RNA/DNA helicase or translocase, that causes dissociation of RNA pol from DNA template and releases RNA. It also has ATPase region.
In rho dependent termination, there are specific sites, transcribed called the rho–dependent terminators. These contain two separate sites; a rho utilization site called the ‘rut site’ and a downstream transcription stop point also called as ‘tsp site’ (see fig 7).
Rho binds RNA at the transcribed ‘rut site’ region. This binding activates RNA-dependent ATPase activity of Rho. Rho moves along the RNA till it reached the RNA pol. The energy derived from the hydrolysis of ATP is used by the rho, with helicase activity, to unwind RNA-DNA hybrid from RNA pol and results into termination (fig 7).
(Just for info: Read more about Rho-dependent termination in a paper.)
In prokaryotes, protein synthesis or the translation starts simultaneously with the transcription, while the mRNA is still being synthesized. This is possible due to the lack of nucleus or any type of compartmentalisation. Hence, the transcript is immediately translated to synthesize the corresponding protein.
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References:
Cooper (2000) The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates.
Guttman (2001) Prokaryotes. Encyclopedia of Genetics. Pg 1549.
Murakami (2015) Structural Biology of Bacterial RNA Polymerase. Biomolecules 5(2): 848–864.
Molecular Cell Biology. 4th edition. Lodish et al. New York: W. H. Freeman; 2000.
Belitsky & Schütz (2018) RNA Polymerase interactions and elongation rate. Published in Journal of theoretical biology. DOI:10.1016/j.jtbi.2018.11.025/
Banerjee et al.(2006) Rho-dependent Transcription Termination: More Questions than Answers. J Microbiol. 44(1): 11–22.
Pelley (2012) RNA Transcription and Control of Gene Expression. Elsevier’s Integrated Review Biochemistry (Second Edition). Chapter 16: 137-147.
Kaplan & Donnell (2003) Rho Factor: Transcription Termination in Four Steps. Current Biology 13(18): R714-716.
Skordalakes & Berger (2003) Structure of the Rho Transcription Terminator: Mechanism of mRNA Recognition and Helicase Loading. Cell 114(1): 135-146.
Weiss et al. (2017) The ω subunit governs RNA Polymerase stability and transcriptional specificity in Staphylococcus aureus. Journal of Bacteriology 199(2):e00459-16
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