Regulation of Gene Expression in Prokaryotes:- Many Genes for Amino Acid Biosynthetic Enzymes Are Regulated by Transcription Attenuation
The 20 common amino acids are required in large amounts for protein synthesis, and E. coli can synthesize all of them. The genes for the enzymes needed to synthesize a given amino acid are generally clustered in an operon and are expressed whenever existing supplies of that amino acid are inadequate for cellular requirements. When the amino acid is abundant, the biosynthetic enzymes are not needed and the operon is repressed. The E. coli tryptophan (trp) operon (Fig. 2) includes five genes for the enzymes required to convert chorismate to tryptophan. Note that two of the enzymes catalyze more than one step in the pathway. The mRNA from the trp operon has a half-life of only about 3 min, allowing the cell to respond rapidly to changing needs for this amino acid. The Trp repressor is a homodimer, each subunit containing 107 amino acid residues (Fig. 1). When tryptophan is abundant it binds to the Trp repressor, causing a conformational change that permits the repressor to bind to the trp operator and inhibit expression of the trp operon. The trp operator site overlaps the promoter, so binding of the repressor blocks binding of RNA polymerase.
Once again, this simple on/off circuit mediated by a repressor is not the entire regulatory story. Different cellular concentrations of tryptophan can vary the rate of synthesis of the biosynthetic enzymes over a 700-fold range. Once repression is lifted and transcription be gins, the rate of transcription is fine-tuned by a second regulatory process, called transcription attenuation, in which transcription is initiated normally but is abruptly halted before the operon genes are transcribed. The frequency with which transcription is attenuated is regulated by the availability of tryptophan and relies on the very close coupling of transcription and translation in bacteria. The trp operon attenuation mechanism uses signals encoded in four sequences within a 162-nucleotide leader region at the 5end of the mRNA, preceding the initiation codon of the first gene (Fig. 2a). Within the leader lies a region known as the attenuator, made up of sequences 3 and 4. These sequences base-pair to form a G≡C-rich stem-and-loop structure closely followed by a series of U residues. The attenuator structure acts as a transcription terminator (Fig. 2b). Sequence 2 is an alternative complement for sequence 3 (Fig. 2c). If sequences 2 and 3 base-pair, the attenuator structure cannot form and transcription continues into the trp biosynthetic genes; the loop formed by the pairing of sequences 2 and 3 does not obstruct transcription.
FIGURE 1 Trp repressor. The repressor is a dimer, with both sub units (gray and light blue) binding the DNA at helix-turn-helix motifs (PDB ID 1TRO). Bound molecules of tryptophan are in red.
Regulatory sequence 1 is crucial for a tryptophan sensitive mechanism that determines whether sequence 3 pairs with sequence 2 (allowing transcription to continue) or with sequence 4 (attenuating transcription). Formation of the attenuator stem-and-loop structure depends on events that occur during translation of regulatory sequence 1, which encodes a leader peptide (so called because it is encoded by the leader region of the mRNA) of 14 amino acids, two of which are Trp residues. The leader peptide has no other known cellular function; its synthesis is simply an operon regulatory device.
FIGURE 2 Transcriptional attenuation in thetrpoperon. Tran scription is initiated at the beginning of the 162-nucleotide mRNA leader encoded by a DNA region called trpL (see Fig. 2). A regulatory mechanism determines whether transcription is attenuated at the end of the leader or continues into the structural genes. (a)The trpm RNA leader (trpL). The attenuation mechanism in the trpoperon involves sequences 1 to 4 (highlighted). (b) Sequence 1 encodes a small peptide, the leader peptide, containing two Trp residues (W); it is translated immediately after transcription begins. Sequences 2 and 3 are complementary, as are sequences 3 and 4. The attenuator structure forms by the pairing of sequences 3 and 4 (top). Its structure and function are similar to those of a transcription terminator (see Fig. 26–7). Pairing of sequences 2 and 3 (bottom) prevents the attenuator structure from forming. Note that the leader peptide has no other cellular function. Translation of its open reading frame has a purely regulatory role that determines which complementary sequences (2 and 3 or 3 and 4) are paired. (c)Base-pairing schemes for the complementary regions of the trpm RNA leader.
This peptide is translated immediately after it is tran scribed, by a ribosome that follows closely behind RNA polymerase as transcription proceeds. When tryptophan concentrations are high, concentrations of charged tryptophan tRNA (Trp-tRNATrp) are also high. This allows translation to proceed rapidly past the two Trp codons of sequence 1 and into sequence 2, before sequence 3 is synthesized by RNA polymerase. In this situation, sequence 2 is covered by the ribosome and unavailable for pairing to sequence 3 when sequence 3 is synthesized; the attenuator structure (sequences 3 and 4) forms and transcription halts (Fig. 2b, top). When tryptophan concentrations are low, however, the ribosome stalls at the two Trp codons in sequence 1, because charged tRNATrp is less available. Sequence 2 remains free while sequence 3 is syn the sized, allowing these two sequences to base-pair and permitting transcription to proceed (Fig. 2b, bot tom). In this way, the proportion of transcripts that are attenuated declines as tryptophan concentration declines. Many other amino acid biosynthetic operons use a similar attenuation strategy to fine-tune biosynthetic en zymes to meet the prevailing cellular requirements. The 15 amino acid leader peptide produced by the phe operon contains seven Phe residues. The leu operon leader peptide has four contiguous Leu residues. The leader peptide for the his operon contains seven contiguous His residues. In fact, in the his operon and a number of others, attenuation is sufficiently sensitive to be the only regulatory mechanism.
