Research on Prokaryotic Transcription

Studies in E. coli primarily led to the discovery of the detailed mechanism of prokaryotic transcription. Overall, it appears that prokaryotic transcription requires the DNA double helix to partially unwind in the region where mRNA synthesis takes place. This region of unwinding is known as the transcription bubble. Also, transcription always proceeds from the same DNA strand for each gene, the template strand. The resulting mRNA product, which is complementary to the template strand, is almost identical to the other DNA strand, called the non-template strand. The only difference is that in mRNA, all of the T nucleotides are replaced with U nucleotides.

To be more specific, prokaryotes lack a membrane-enclosed nucleus. Therefore, the processes of transcription, translation, and mRNA degradation can all occur simultaneously. Prokaryotic transcription is initiated, as in all species, with the binding of the RNA polymerase complex to a special DNA sequence at the beginning of the gene known as the promoter. RNA polymerase essentially catalyzes the polymerization of ribonucleoside 5'-triphosphates (NTPs) as directed by a DNA template in the 5' to 3' direction. Furthermore, RNA polymerase (in E.coli particularly) is composed of five polypeptide subunits, two of which are identical. Four of these subunits, a, a, and W make up the polymerase core enzyme. These subunits assemble every time a gene is transcribed, and they disassemble once transcription is complete. Each subunit has a unique role; the two a-subunits are necessary to assemble the polymerase on the DNA; the ¬ subunit binds to the ribonucleoside triphosphate that will become part of the nascent "recently born" mRNA molecule, and theW binds the DNA template strand. The fifth subunit, o, is involved only in transcription initiation. The o subunit provides transcriptional specificity such that the polymerase begins to synthesize mRNA from an appropriate initiation site. Hence, without o, the core enzyme would transcribe from random sites which is problematic since the synthesis of a functional RNA must start at the beginning of a gene.

Moreover, as pointed out earlier, in order to initiate transcription of a gene RNA polymerase must bind to a DNA sequence called the promoter. In most cases, promoters exist upstream of the genes they regulate. It appears that the specific sequence of a promoter is very important because it determines whether the corresponding gene is transcribed all the time, often, or infrequently. However, promoters may vary among prokaryotic genomes, still few elements tend to be conserved. For instance, at the -10 and -35 regions upstream of the initiation site +1, reside two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species. The -10 consensus sequence is TATAAT; and the -35 sequence, TTGACA, is recognized and bound by o. Once this interaction is made, the subunits of the core enzyme, RNA polymerase, bind to the site. The A-T-rich -10 regions are known to facilitate unwinding of the DNA template.

In addition, transcription initiation is followed by elongation of the transcript, and termination. The transcription elongation phase usually begins with the release of the o subunit from the polymerase. The dissociation of o allows the core enzyme to proceed along the DNA template. As elongation proceeds, the DNA is continuously unwound ahead of the core enzyme and rewound behind it. Also, because the base pairing between DNA and RNA is not stable enough to maintain the stability of the mRNA components, RNA polymerase acts as a stable linker between the DNA template and the nascent RNA strands to ensure that elongation is not interrupted prematurely. Once a gene is transcribed, termination signals instruct the prokaryotic polymerase to dissociate from the DNA template and liberate the newly made mRNA. There are two kinds of termination signals depending on the gene being transcribed: the protein-based and the RNA-based signal. Upon termination, the process of transcription is complete.