Initiation of Transcription in Prokaryotes

Prokaryotic cells contain a single type of RNA polymerase. The sigma factor enables RNA polymerase to begin transcription at specific sites. The sigma factor may also promote melting of the DNA strands of the promoter DNA helix. Once transcription has begun, the sigma subunit dissociates from the DNA template.

Prokaryotic Promoters

Bacterial promoters are located immediately adjacent to the transcription initiation site.

There are two consensus sequences in prokaryotic promoters:

• One is 35 base pairs (bp) upstream from the initiation site and typically occurs as the sequence TTGACA. This site is known as the -35 region, and it is the sequence recognized by the sigma factor.
• The second consensus sequence is about 10 bp upstream (or -10bp) from the initiation site of transcription and occurs as the consensus sequence TATAAT. It is known as the Pribnow box, and is responsible for identifying the precise nucleotide at which transcription begins.
• Certain base substitutions in both the -35 and -10 regions can greatly affect the rate of transcription. Thus promoters are more than simply recognition regions: they can act as important control sites in regulating the rate of gene expression.

Termination of transcription

Transcription terminates at specific nucleotide sequences.

• In some cases, a protein called the rho factor is required for termination
• Most of the times, however, the termination of transcription is signaled within the RNA being synthesized by a GC-rich inverted repeat followed by four U residues.

Operons

A bacterial cell lives in direct contact with its environment. In bacteria, the genes that encode the enzymes of a metabolic pathway are usually clustered together on the chromosome in a functional complex called an operon

A typical bacterial operon consists of structural genes, a promoter region, an operator region, and a regulatory gene.

Structural genes code for the enzymes themselves. RNA polymerase transcribes all of the genes into a single mRNA.

The promoter is the site where the RNA polymerase binds to the DNA prior to beginning transcription.

The operator serves as the binding site for the protein called the repressor.

The regulatory gene encodes the repressor protein.

The lac Operon is an example of an inducible operon

An inducible operon is one in which the presence of a key metabolic substance (e.g. lactose) induces transcription of the structural genes. Thus lactose is the inducer.

The lac operon contain three tandem structural genes:

z gene encodes beta-galactosidase which cleaves lactose

y gene encodes galtgactoside permease which promotes the entry of lactose into the cell

a gene encodes a transacetylase that adds an acetyl group to lactose as it is tyaken up by the cell.

The lac Operon is under negative control

• In an inducible operon, such as the lac operon, the repressor protein, encoded by the regulatory gene, is able to bind to the DNA only in the absence of lactose (the inducer). The repressor exerts negative control by preventing the initiation of transcription and therefore gene expression.

The lac Operon is also under positive control. This positive control is by cyclic adenosine monophosphate (cAMP).

• If bacterial cells are supplied with glucose as well as lactose or galactose, the cells catabolize the glucose and ignore the other compounds. The glucose suppresses the production of various catabolic enzymes, such as beta-galactosidase.
• The concentration of cAMP in the cells is inversely related to the glucose concentration (the higher the glucose concentration, the lower the cAMP concentration)
• cAMP acts by binding to cAMP receptor protein (CRP)
• By itself, CRP is unable to bind to DNA. However the cAMP-CRP complex binds to the lac control region.
• Thus, the presence of the bound cAMP-CRP complex is necessary for the initiation of transcription of the operon, even when lactose is present and the repressor is inactivated.

The Trp Operon is an example of a repressible operon

• The repressor is unable to bind to the operator DNA by itself. Instead, the repressor can only bind to the DNA if the repressor is complexed to a specific factor, such as tryptophan. Tryptophan acts as a co-repressor.
• In the absence of tryptophan, the operator site is open to binding by RNA polymerase and transcription of enzymes necessary for the synthesis of tryptophan.
• Although the trp operon is regulated in part by a repressor, an additional level of control is provided by transcriptional attenuation. The site of attenuation is located 162 nucleotides downstream of the transcription start site. If tryptophan is abundant, most transcription terminates at this site; only if tryptophan is scarce does transcription continue to yield functional Trp mRNA. The mechanism of attenuation depends on the fact that translation in bacteria is coupled with transcription, so ribosomes begin translating the 5' end of an mRNA while it is still being synthesized.

Tools in Molecular Biology

Gel Electrophoresis

Electrophorectic-mobility shift Assay

DNA footprinting

DNA sequencing

DNA cloning

Polymerase Chain Reaction (PCR)

Column Chromatography