Also in prokaryotes ... tryptophan operan

          The trp Operon

The trp operon (see diagram below) encodes the genes for the synthesis of tryptophan. This cluster of genes, like the lac operon, is regulated by a repressor that binds to the operator sequences. The activity of the trp repressor for binding the operator region is enhanced when it binds tryptophan; in this capacity, tryptophan is known as a corepressor. Since the activity of the trp repressor is enhanced in the presence of tryptophan, the rate of expression of the trp operon is graded in response to the level of tryptophan in the cell.

Expression of the trp operon is also regulated by attenuation. The attenuator region, which is composed of sequences found within the transcribed RNA, is involved in controlling transcription from the operon after RNA polymerase has initiated synthesis. The attenuator of sequences of the RNA are found near the 5' end of the RNA termed the leader region of the RNA. The leader sequences are located prior to the start of the coding region for the first gene of the operon (the trpE gene). The attenuator region contains codons for a small leader polypeptide, that contains tandem tryptophan codons. This region of the RNA is also capable of forming several different stable stem-loop structures.

Depending on the level of tryptophan in the cell and hence the level of charged trp-tRNAs, the position of ribosomes on the leader polypeptide and the rate at which they are translating allows different stem-loops to form. If tryptophan is abundant, the ribosome prevents stem-loop 1-2 from forming and thereby favors stem-loop 3-4. The latter is found near a region rich in uracil and acts as the transcriptional terminator loop as described in the RNA synthesis page. Consequently, RNA polymerase is dislodged from the template.

The operons coding for genes necessary for the synthesis of a number of other amino acids are also regulated by this attenuation mechanism. It should be clear, however, that this type of transcriptional regulation is not feasible for eukaryotic cells.

Regulation of the trp operon in E. coli. The trp operon is controlled by both a repressor protein binding to the operator region as well as by translation-induced transcriptional attenuation. The trp repressor binds the operator region of the trp operon only when bound to tryptophan. This makes tryptophan a co-repressor of the operon. The trpL gene encodes a non-functional leader peptide which contains several adjacent trp codons. The tructural genes of the operon responsible for tryptophan biosynthesis are trpE, D, C, B and A. When trptophan level are high some binds to the repressor which then binds to the operator region and inhibits transcription. The mechanism of attenuation of the trp operon is diagrammed below.

Attenuation of the trp operon. The attenuation region of the trp operon contains sequences that allow the resulting mRNA to form several different stem-loop structures. These regions are identified as 1 through 4. The stem-loops that are significant as to whether transcription is attenuated or not are formed between regions 2 and 3 or between regions 3 and 4. When tryptophan levels are high there is plenty of charged trp-tRNAs available and ribosomes translating the leader peptide encoded by the trpL gene do not stall at the repeated trp codons in the leader peptide. Under these conditions the ribosomes rapidly cover regions 1 and 2 of the mRNA which allows the stem-loop composed of regions 3 and 4 to form. The stem-loop formed by regions 3-4 results in a transcriptional termination structure and transcription of the trp operon ceases, i.e. is attenuated. Conversely, when tryptophan levels are low the level of charged trp-tRNAs will also be low. This leads to a stalling of the ribosomes within the leader peptide when they encounter the trp codon repeats. The ribosome stalls over region 1 of the mRNA which allows step-loop 2-3 to form and prevents the transcriptional termination stem-loop 3-4 from forming. Th

Gene Regulation in prokaryotes -- the lac operan

            The lac Operon

The lac operon (see diagram below) consists of one regulatory gene (the i gene) and three structural genes (z, y, and a). The i gene codes for the repressor of the lac operon. The z gene codes for β-galactosidase (β-gal), which is primarily responsible for the hydrolysis of the disaccharide, lactose into its monomeric units, galactose and glucose. The y gene codes for permease, which increases permeability of the cell to β-galactosides. The a gene encodes a transacetylase. During normal growth on a glucose-based medium, the lac repressor is bound to the operator region of the lac operon, preventing transcription. However, in the presence of an inducer of the lac operon, the repressor protein binds the inducer and is rendered incapable of interacting with the operator region of the operon. RNA polymerase is thus able to bind at the promoter region, and transcription of the operon ensues. The lac operon is repressed, even in the presence of lactose, if glucose is also present. This repression is maintained until the glucose supply is exhausted. The repression of the lac operon under these conditions is termed catabolite repression and is a result of the low levels of cAMP that result from an adequate glucose supply. The repression of the lac operon is relieved in the presence of glucose if excess cAMP is added. As the level of glucose in the medium falls, the level of cAMP increases. Simultaneously there is an increase in inducer binding to the lac repressor. The net result is an increase in transcription from the operon. The ability of cAMP to activate expression from the lac operon results from an interaction of cAMP with a protein termed CRP (for cAMP receptor protein). The protein is also called CAP (for catabolite activator protein). The cAMP-CRP complex binds to a region of the lac operon just upstream of the region bound by RNA polymerase and that somewhat overlaps that of the repressor binding site of the operator region. The binding of the cAMP-CRP complex to the lac operon stimulates RNA polymerase activity 20-to-50-fold.

Regulation of the lac operon in E. coli. The repressor of the operon is synthesized from the i gene. The repressor protein binds to the operator region of the operon and prevents RNA polymerase from transcribing the operon. In the presence of an inducer (such as the natural inducer, allolactose) the repressor is inactivated by interaction with the inducer. This allows RNA polymerase access to the operon and transcription proceeds. The resultant mRNA encodes the β-galactosidase, permease and transacetylase activities necessary for utilization of β-galactosides (such as lactose) as an energy source. The lac operon is additionally regulated through binding of the cAMP-receptor protein, CRP (also termed the catabolite activator protein, CAP) to sequences near the promoter domain of the operon. The result is a 50 fold enhancement of polymerase activity.

Gene Control in Prokaryotes

Gene Control in Prokaryotes

In bacteria, genes are clustered into operons: gene clusters that encode the proteins necessary to perform coordinated function, such as biosynthesis of a given amino acid. RNA that is transcribed from prokaryotic operons is polycistronic a term implying that multiple proteins are encoded in a single transcript.

In bacteria, control of the rate of transcriptional initiation is the predominant site for control of gene expression. As with the majority of prokaryotic genes, initiation is controlled by two DNA sequence elements that are approximately 35 bases and 10 bases, respectively, upstream of the site of transcriptional initiation and as such are identified as the -35 and -10 positions. These 2 sequence elements are termed promoter sequences, because they promote recognition of transcriptional start sites by RNA polymerase. The consensus sequence for the -35 position is TTGACA, and for the -10 position, TATAAT. (The -10 position is also known as the Pribnow-box.) These promoter sequences are recognized and contacted by RNA polymerase.

The activity of RNA polymerase at a given promoter is in turn regulated by interaction with accessory proteins, which affect its ability to recognize start sites. These regulatory proteins can act both positively (activators) and negatively (repressors). The accessibility of promoter regions of prokaryotic DNA is in many cases regulated by the interaction of proteins with sequences termed operators. The operator region is adjacent to the promoter elements in most operons and in most cases the sequences of the operator bind a repressor protein. However, there are several operons in E. coli that contain overlapping sequence elements, one that binds a repressor and one that binds an activator.

As indicated above, prokaryotic genes that encode the proteins necessary to perform coordinated function are clustered into operons. Two major modes of transcriptional regulation function in bacteria (E. coli) to control the expression of operons. Both mechanisms involve repressor proteins. One mode of regulation is exerted upon operons that produce gene products necessary for the utilization of energy; these are catabolite-regulated operons. The other mode regulates operons that produce gene products necessary for the synthesis of small biomolecules such as amino acids. Expression from the latter class of operons is attenuated by sequences within the transcribed RNA.

A classic example of a catabolite-regulated operon is the lac operon, responsible for obtaining energy from β-galactosides such as lactose. A classic example of an attenuated operon is the trp operon, responsible for the biosynthesis of tryptophan.