MtrB from Bacillus subtilis binds specifically to trp leader RNA in a tryptophan-dependent manner.

Abstract

MtrB regulates transcription attenuation of the Bacillus subtilis typ operon. We have shown that MtrB, either from B. subtdis or overexpressed in Escherichwa cohl, binds specifically to RNA from the leader region of the tqp operon by a gel mobility-shift assay. This binding is tryptophan dependent. MtrB binds to a transcript terminated at the trp attenuator (-2 to +138) or a read-through transcript (-2 to +318). MtrB does not bind antisense tbp leader RNA or single-stranded trp leader DNA. These results support the model in which attenuation is controlled by tryptophanactivated MtrB influencing the secondary structure of the leader region transcript to form a terminator structure.An unusual form of attenuation is believed to be the sole means of regulating transcription of the Bacillus subtilis tryptophan (trpEDCFBA) operon in response to L-tryptophan (1). Two alternative RNA secondary structures, which form in the 5' leader region RNA transcript, control transcription of the operon (1, 2). In the presence of tryptophan, a terminator structure forms, halting transcription in the leader region, whereas in the absence of tryptophan, an antiterminator structure forms, allowing transcription of the operon. The unusual aspect of this attenuation mechanism is that selection between the alternative RNA structures is mediated by a tryptophan-activated trans-acting regulatory factor rather than by synthesis of a leader peptide, such as occurs in the Escherichia coli trp operon. This regulator appears to function by binding to a specific target in the antiterminator RNA stem and, when bound, to promote formation of the transcription terminator structure (1, 2). The product of the methyltryptophan-resistance (mtr) locus is thought to be the regulatory factor because mutations in this locus eliminate control of the trp operon, resulting in constitutive production of the tryptophan biosynthetic enzymes (1-3). The mtr locus from B. subtilis has been cloned and sequenced and is a two-gene operon consisting of mtrA and mtrB (4). Recently, MtrA (22 kDa) has been identified as GTP cyclohydrolase I, and MtrB (8 kDa) has been shown to be the regulator of the trp operon (5).Evidence that the regulator interacts with an RNA target first came from in vivo studies in which overexpressing the trp leader RNA transcript in trans resulted in constitutive expression of the trp operon, presumably by titrating the regulatory MtrB factor (1, 2). Multiple copies of the trp leader as DNA did not affect expression of the trp operon, which indicated that the regulator target is RNA. Deletion analysis of the leader region localized the regulator target to a 10-base sequence (AGAAUGAGUU), which is repeated twice in the leader transcript, including once in the antiterminator stem (2). Deleting either of these sites lessened effectiveness of the region as a target and deleting both repeats completely abolished target function.The current model for attenuation control of the B. subtilis trp operon suggests that tryptophan-activated MtrB binds t...

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