Regulation of Prokaryotic Gene Expression

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Chapter: Biochemistry : Regulation of Gene Expression

In prokaryotes such as Escherichia coli (E. coli), regulation of gene expression occurs primarily at the level of transcription and, in general, is mediated by the binding of trans-acting proteins to cis-acting regulatory elements on their single DNA molecule (chromosome).


REGULATION OF PROKARYOTIC GENE EXPRESSION

In prokaryotes such as Escherichia coli (E. coli), regulation of gene expression occurs primarily at the level of transcription and, in general, is mediated by the binding of trans-acting proteins to cis-acting regulatory elements on their single DNA molecule (chromosome). [Note: Regulating the first step in the expression of a gene is an efficient approach, insofar as energy is not wasted making unneeded gene products.] Transcriptional control in prokaryotes can involve the initiation or premature termination of transcription.

 

A. Transcription of messenger RNA from bacterial operons

In bacteria, the structural genes that code for proteins involved in a particular metabolic pathway are often found sequentially grouped on the chromosome along with the cis-acting regulatory elements that determine the transcription of these genes. The transcription product is a single polycistronic messenger RNA (mRNA). The genes are, thus, coordinately controlled (that is, turned on or off as a unit). This entire package is referred to as an operon.

 

B. Role of operators in prokaryotic transcription

Prokaryotic operons contain an operator, a segment of DNA that regulates the activity of the structural genes of the operon. If the operator is not bound by a repressor molecule, RNA polymerase passes over the operator and reaches the protein-coding genes which it transcribes to mRNA. If a repressor molecule is bound to the operator, the polymerase is blocked and does not produce mRNA. As long as the repressor is bound to the operator, no proteins are made. However, when an inducer molecule is present, it binds to the repressor, causing the repressor to change shape so that it no longer binds the operator. When this happens, the RNA polymerase can proceed with transcription. One of the best-understood examples is the inducible lactose operon of E. coli that illustrates both positive and negative regulation (Figure 32.4).


Figure 32.4 The lactose operon of E. coli. *[Note: Even when the operon has been turned off by catabolite repression, the repressor transiently dissociates from the operator at a slow rate, allowing a very low level of expression. The synthesis of a few molecules of permease (and β-galactosidase) allows the organism to respond rapidly should glucose become unavailable.] CAP = catabolite activator protein; cAMP = cyclic adenosine monophosphate; mRNA = messenger RNA.

 

C. The lactose operon

The lactose (lac) operon contains the genes that code for three proteins involved in the catabolism of the disaccharide lactose: The lacZ gene codes for β-galactosidase, which hydrolyzes lactose to galactose and glucose; the lacY gene codes for a permease, which facilitates the movement of lactose into the cell; and the lacA gene codes for thiogalactoside transacetylase, which acetylates lactose. [Note: The physiologic function of this acetylation is unknown.] All of these proteins are maximally produced only when lactose is available to the cell and glucose is not. [Note: Bacteria use glucose, if available, as a fuel in preference to any other sugar.] The regulatory portion of the operon is upstream of the three structural genes and consists of the promoter region where RNA polymerase binds and two additional sites, the operator (O) site and the CAP site, where regulatory proteins bind. The lacZ, lacY, and lacA genes are expressed only when the O site is empty, and the CAP site is bound by a complex of cyclic adenosine monophosphate ([cAMP]) and the catabolite activator protein (CAP), sometimes called the cAMP regulatory protein (CRP). A regulatory gene, the lacI gene, codes for the repressor protein (a trans-acting factor) that binds to the O site with high affinity. [Note: The lacI gene has its own promoter.]

 

1. When only glucose is available: In this case, the lac operon is repressed (turned off). Repression is mediated by the repressor protein binding via a helix-turn-helix motif (Figure 32.5) to the operator site, which is downstream of the promoter region (see Figure 32.4A). Binding of the repressor interferes with the progress of RNA polymerase and blocks transcription of the structural genes. This is an example of negative regulation.


Figure 32.5 Helix-turn-helix motif of the lac repressor protein.

 

2. When only lactose is available: In this case, the lac operon is induced (maximally expressed, or turned on). A small amount of lactose is converted to an isomer, allolactose. This compound is an inducer that binds to the repressor protein, changing its conformation so that it can no longer bind to the operator. In the absence of glucose, adenylyl cyclase is active, and sufficient quantities of cAMP are made and bind to the CAP protein. The cAMP–CAP trans-acting complex binds to the CAP site, causing RNA polymerase to more efficiently initiate transcription at the promoter site (see Figure 32.4B). This is an example of positive regulation. The transcript is a single polycistronic mRNA molecule that contains three sets of start and stop codons. Translation of the mRNA produces the three proteins that allow lactose to be used for energy production by the cell. [Note: In contrast to the inducible lacZ, lacY, and lacA genes, whose expression is regulated, the lacI gene is constitutive. Its gene product, the repressor protein, is always made and is active unless the inducer is present.]

 

3. When both glucose and lactose are available: In this case, transcription of the lac operon is negligible, even if lactose is present at a high concentration. Adenylyl cyclase is inhibited in the presence of glucose (a process known as catabolite repression) so no cAMP–CAP complex forms, and the CAP site remains empty. RNA polymerase is, therefore, unable to effectively initiate transcription, even though the repressor may not be bound to the operator region. Consequently, the three structural genes of the operon are not expressed (see Figure 32.4C).

 

D. Tryptophan operon

The tryptophan (trp) operon contains five structural genes that code for enzymes required for the synthesis of the amino acid, tryptophan. As with the lac operon, the trp operon is subject to negative control. However, for the repressible trp operon, negative control includes Trp itself binding to a repressor protein and facilitating the binding of the repressor to the operator: Trp is a corepressor. Because repression by Trp is not always complete, unlike the lac operon, the trp operon is also regulated by a process known as attenuation. With attenuation, transcription is initiated but is terminated well before completion (Figure 32.6). If Trp is plentiful, transcription initiation that escaped repression by Trp is attenuated (stopped) by the formation at the 5I -end of the mRNA of a hairpin (stem–loop) structure like that seen in rho-independent termination. [Note: Transcription and translation are temporally linked in prokaryotes, and, therefore, attenuation also results in the formation of a truncated, nonfunctional peptide product that is rapidly degraded.] If Trp becomes scarce, the operon is expressed. The 5I -end of the mRNA contains two adjacent codons for Trp. The lack of Trp causes ribosomes to stall at these codons, covering regions of the mRNA required for formation of the attenuation hairpin. This prevents attenuation and allows transcription to continue.


Figure 32.6 Attenuation of transcription of the trp operon when tryptophan is plentiful. mRNA = messenger RNA.

Transcriptional attenuation can occur in prokaryotes because translation of an mRNA begins before its synthesis is complete. This does not occur in eukaryotes because the presence of a membrane-bound nucleus spatially and temporally separates transcription and translation.

 

E. Coordination of transcription and translation in prokaryotes

Whereas transcriptional regulation of mRNA production is primary in bacteria, regulation at the level of ribosomal RNA (rRNA) and protein synthesis also occurs and plays important roles in the microbe’s ability to adapt to environmental stress.

 

1. Stringent response: E. coli has seven operons that synthesize the rRNA needed for ribosome assembly, and each is regulated in response to changes in environmental conditions. Regulation in response to amino acid starvation is known as the stringent response. The binding of an uncharged transfer RNA (tRNA) to the A site of a ribosome triggers a series of events that leads to the production of a polyphosphorylated guanosine, ppGpp. The synthesis of this unusual derivative of guanosine diphosphate (GDP) is catalyzed by stringent factor (RelA), an enzyme physically associated with ribosomes. Elevated levels of ppGpp result in inhibition of rRNA synthesis (Figure 32.7). [Note: In addition to rRNA, tRNA synthesis and some mRNA synthesis (for example, for ribosomal proteins) are also inhibited. However, synthesis of mRNAs for enzymes required for amino acid biosynthesis is not inhibited. ppGpp appears to alter promoter selection through use of different sigma-factors for RNA polymerase.]


Figure 32.7 Regulation of transcription by the stringent response to amino acid starvation. S = Svedberg unit.

 

2. Regulatory ribosomal proteins: Operons for ribosomal proteins (r-proteins) can be inhibited by an excess of their own protein products. For each operon, one specific r-protein functions in the repression of translation of the polycistronic mRNA from that operon (Figure 32.8). The r-protein does so by binding to the Shine-Dalgarno (SD) sequence located on the mRNA just upstream of the first initiating AUG codon and acting as a physical impediment to the binding of the small ribosomal subunit to the SD sequence. One r-protein thus inhibits synthesis of all the r-proteins of the operon. This same r-protein also binds to rRNA and with a higher affinity than for mRNA. If the concentration of rRNA falls, the r-protein then is available to bind its own mRNA and inhibit its translation. This coordinated regulation keeps the synthesis of r-proteins in balance with the transcription of rRNA, so that each is present in appropriate amounts for the formation of ribosomes.


Figure 32.8 Regulation of translation by an excess of ribosomal proteins. mRNA = messenger RNA; rRNA = ribosomal RNA; r-protein = ribosomal protein.

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