Textbook assignment: Chapter 18, pages 529-534.
Major topics:
Diversity of transcriptional controls in prokaryotic cells: This is the second of three scheduled lectures that explore the diverse ways in which transcription can be controlled in prokaryotic cells. In the previous lecture, the operon concept was introduced and illustrated in terms of positive control of the lac operon by lactose (via allolactose) and negative control by glucose (achieved indirectly by reversing a positive control system involving the cAMP/CAP complex). This lecture begins with a brief review of the lac -operon, and a brief examination of the combination of positive and negative controls in the arabinose operon. It then focuses on the tryptophan (trp ) operon to demonstrate end product-activated repression of operons coding for enzymes involved in biosynthetic pathways, as well as a novel leader-attenuator mechanism that is used to fine tune biosynthetic levels. The next lecture will explore some of the ways in which bacterial viruses take over the transcriptional machinery of their host cells, as well as the molecular switches involved in the choice between lytic infection and lysogeny in bacteriophage lambda.
Review of lac operon: For transcription of the three genes in the lac operon to occur at more than a low constituitive level, two conditions must be met: 1) glucose must be unavailable as an energy source; and 2) lactose must be available. Glucose keeps cAMP levels low in the cell. When glucose becomes scarce, cAMP levels rise. The cAMP forms a complex with the catabolite activator protein (CAP), which interacts with the promoter of the lac operon to increase transcription. However, in the absence of lactose, the lac repressor protein still occupies the operator site and keeps the transcriptional level low. If lactose is available, a small amount of it is converted to allolactose, which binds to the repressor protein and causes it to dissociate from the operator site. This allows a high level of transcription of the lac operon to occur, but only when there is no glucose and the cAMP-CAP complex has activated the promoter. This system allows the cells to save energy by turning on expression of the genes needed for utilization of a second choice substrate only when the first choice (glucose) is not available and the second choice (lactose) is available.
The arabinose operon: Arabinose is another type of sugar that is used only when glucose is not available. The arabinose operon has a CAP/cAMP control similar to that of the lactose operon. In addition, expression of the genes needed for arabinose utilization is induced only when arabinose is present. In this case, the regulatory protein that interacts with arabinose has a dual role. In the absence of arabinose, it binds to the operator and prevents transcription of the operon. When arabinose is present and bound to the regulatory protein, the operator site is no longer blocked. In addition, the complex of the regulatory protein and arabinose interacts with a site adjacent to the operator to increases the level of transcription above the basal level that occurs in the complete absence of the regulatory protein (see page 529 and figure 18.7 in the textbook.
The tryptophan operon: The trp operon contains the structural genes for five enzymes that are required for the biosynthesis of tryptophan. These enzymes are needed only when tryptophan is not available from the extracellular environment. Coordinated transcription of these five genes is controlled in two very different ways: 1) through the use of an end-product activated repressor protein; and 2) through the use of a novel leader-attenuator mechanism. The operon itself consists of a promoter, an operator site, the leader-attenuation site (described later), and the five structural genes. The trp repressor protein is coded at a separate site.
End product-mediated repression of the trp operon: Repression of the trp operon utilizes an operator site and a repressor protein whose overall functions resemble those of the lac operator and repressor. However, by itself, the trp repressor protein is inactive. For repression to occur, tryptophan must be present at a sufficiently high concentration to bind to the inactive repressor protein and convert it to an active repressor that will bind to the operator and stop transcription. Thus, tryptophan serves as a corepressor. When the environment contains enough tryptophan, the operon is repressed, allowing the cell to save the energy that would otherwise be expended for the transcription and translation of the five gene products coded for in the operon. .
Derepression: When the level of available tryptophan drops, mass action causes it to dissociate from the repressor protein, which then becomes non-functional. This causes derepression to occur, giving the cell the capability of synthesizing its own tryptophan. Thus absence of the corepressor (tryptophan) inactivates a negative control, rather than directly stimulating transcription. This arrangement makes it relatively easy for the cells to respond quickly to the absence of tryptophan by derepressing expression of genes whose function is needed only for the synthesis of tryptophan. The lac operon is also activated by derepression, but the mechanism is exactly the opposite from that for the trp operon. The lac repressor alone is active, but is deactivated by allolactose, whereas the trp repressor alone is inactive, but is activated by tryptophan.
Attenuation of transcription in the trp operon: The attenuation mechanism is described in detail in the textbook. It allows the extent of transcription of the trp operon to be modified in response to the amount of charged trp- tRNA in the cell, thus providing a more sensitive index of when there is enough tryptophan present for protein synthesis. In brief summary, the first part of the trp transcript includes four regions (1-4) that can form two stem loops by pairing 1:2 and 3:4, or a single stem loop by pairing 2:3, with regions 1 and 4 unpaired. Immediately following region 4, there is a sequence of seven uracils (U). If the 3:4 stem loop is allowed to form as regions 3 and 4 are being transcribed, that loop and the oligo-U sequence that is synthesized immediately afterward generate a prokaryotic transcription termination signal. This causes transcription to stop before it reaches the structural genes for the five enzymes coded by the trp operon, which begin just downstream from the oligo-U sequence. The leader-attenuator system operates by controlling the extent of formation of the 3:4 loop, and thus of the termination signal, as described below.
Ribosomal stalling due to insufficient charged tryptophane-tRNA: There is an open reading frame for a short peptide of 14 amino acids, with its translational initiation site just upstream from region 1. The coding sequence includes two trp codons side by side in positions 8 and 9, located at the start of region 1. Translation starts soon after the free 5'-end of the mRNA leaves the RNA polymerase. If tryptophan levels inside the cell are low, charged trp-tRNA will be in short supply and the ribosome will temporarily stall at the two trp codons in region 1. This will prevent region 1 from forming a stem loop with region 2, which will encourage regions 2 and 3 to form a stem loop. This in turn keeps regions 3 and 4 from forming the stem-loop plus oligo-U termination signal, allowing the RNA polymerase to move beyond the attenuator site and begin to transcribe the structural genes.
Attenuation when adequate trp-tRNA is present: On the other hand, if there is enough intracellular trp-tRNA so the ribosome can move quickly past the two trp codons, the remainder of the short (14 amino acid) leader peptide is synthesized and the ribosome departs form the transcript. This allows region 1, which no longer contains a ribosome to pair with region 2, leaving region 3 available to pair with region 4 and thus to form the stem-loop termination signal before the RNA polymerase has left the area. This results in early termination of transcription before any of the structural genes are transcribed. In addition, the relative probabilities of continuing transcription vs. terminating it are highly responsive to small variations in the intracellular concentration of charged trp-tRNA, providing a much more sensitive control system than can be achieved by interaction of free tryptophan with the trp repressor protein as the only control mechanism.
Other attenuation systems: A similar combination of end product-mediated repression and leader attenuation is also used for operons encoding enzymes for synthesis of a number of other amino acids in various bacteria. In a more extreme case, an operon encoding nine enzymes for the biosynthesis of histidine in Salmonella typhimurium is controlled entirely by a leader-attenuation mechanism, with no repressor protein at all.