The mechanism and control of DNA transcription
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Transcription in Prokaryotes

A transcription unit comprises the DNA between a promoter, where transcription initiates, and a terminator, where it ends. One strand of the DNA in the region serves as a template for the synthesis of a complementary strand of RNA. The RNA-DNA hybrid is very short (2-3 bp) and transient, as the transcription “bubble” moves along DNA.

The RNA polymerase holoenzyme that synthesises bacterial RNA can be separated into two components. Core enzyme is a multimer having the subunits beta, beta prime and two alpha, that is responsible for elongating the RNA chain. Sigma factor is a single subunit that is required at the stage of initiation for recognising the promoter. Core enzyme has a general affinity for DNA. The addition of sigma factor reduces the affinity of the enzyme for nonspecific binding to DNA, but increases its affinity for promoters. The rate at which RNA polymerase finds its promoters is too great to be accounted for by diffusion and random contacts with DNA: direct exchange of DNA sequences held by the enzyme may be involved. Bacterial RNA polymerases can produce polysistronic mRNA, that is one section of RNA that can code for more than one protein.

E-coli promoter sites are identified by two short “consensus sequences” at –35 and –10 positions upstream of the initiation site (+1) that are conserved during footprinting experiments. The promoters have sequences very similar to the consensus sequences. The sequence at –35 is --TTGACA-- and is called the “–35 refion”. The sequence at

-10 is --TATAAT—and is called the “Pribnow bow”. The sigma 70 subunit binds to these promoter sites by covalent bonds. The initiation site usually has an AG base pair. The nucleoside at the initiation site retains its 5 prime triphosphate.

Bacterial RNA polymerase terminates transcription at two types of sites. Intrinsic terminators contain a G-C rich hairpin followed by a run of U residues. The are recognised in vitro by core enzyme alone. Rho dependant terminators require rho factor both in vitro and in vivo; they have a stretch of 50-90 nucleotides preceding the site of termination that is rich in C and poor in G residues. Rho factor is an essential protein that acts as an ancillary termination factor, which recognises RNA and acts at sites where RNA polymerase has paused. The termination activity requires ATP hydrolysis. The primary transcript has “ragged ends”; this means there is no specific termination signal. Post transcriptional processing occurs, a signal within the sequence causes cleavage of the very end of the RNA, then a poly-A tail is added by poly-A polymerase. The adenosine for this comes from ATP.

Transcription in Eukaryotes

Overall the process is very similar to that in prokaryotes, except that eukaryotes have monosistronic RNA. Eukaryotes are also different in that they have various types of RNA polymerase.

·        Polymerase I makes RNA and is nucleoplasmic.

·        Polymerase II makes rRNA and is nucleolar.

·        Polymerase III makes mRNA and is nucleoplasmic.

Some genes, e.g. the clotting factor VIII have many introns across the gene – 25 introns = 177000bp to 9000bp of exons.

The lac operon

The lac operon contains three genes, lac Z, lac Y and lac A. lac Z codes for Beta-galactosidase. lac Y codes for lactose permease, which spans the E-coli membrane and uses an electrochemical gradient to pump lactose into the cell.

All three genes are coordinately regulated. When glucose is the energy source there is a very low rate of synthesis from the lac operon. With lactose as the main energy source there is a very high rate of synthesis from the lac operon.

Comparing the sequences of wild type cells with operative and promoter mutants has identified the lac operon control sites. Further analysis of the lac control region has been carried out using DNase1 footprinting techniques. These experiments have shown that the lac operon contains binding sites for three proteins; RNA polymerase, lac repressor, and a protein called CAP complexed to cAMP.

The dependence of expression of the lac genes on the concentration of lactose compared to glucose was proven by an experiment involving bacteriophage. Radiolabeled RNA was hybridised with bacteriophage DNA containing the lac operon. Unhybridised RNA was digested. Cells grown in lactose were found to contain lots of labeled RNA, wheras those grown on glucose had a very low content of labeled RNA.

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