TRAP-5′ stem–loop interaction increases the efficiency of transcription termination in the Bacillus subtilis trpEDCFBA operon leader region

Abstract

TRAP regulates expression of the Bacillus subtilis trpEDCFBA operon by a transcription attenuation mechanism in which tryptophan-activated TRAP binds to 11 (G/U)AG repeats in the nascent trp leader transcript. Bound TRAP blocks formation of an antiterminator structure and allows formation of an overlapping intrinsic terminator upstream of the trp operon structural genes. A 59 stem-loop (59SL) structure located upstream of the triplet repeat region also interacts with TRAP. TRAP-59SL RNA interaction participates in the transcription attenuation mechanism by preferentially increasing the affinity of TRAP for the nascent trp leader transcript during the early stages of transcription, when only a few triplet repeats have been synthesized. Footprinting assays indicated that the 59SL contacts TRAP through two discrete groups of single-stranded nucleotides that lie in the hairpin loop and in an internal loop. Filter binding and in vivo expression assays of 59SL mutants established that G7, A8, and A9 from the internal loop, and A19 and G20 from the hairpin loop are critical for proper 59SL function. These nucleotides are conserved among certain other 59SL-containing organisms. Single-round transcription results indicated that the 59SL increases the termination efficiency when transcription is fast; however, the influence of the 59SL was lost when transcription was slowed by reducing the ribonucleoside triphosphate concentration. Since there is a limited amount of time for TRAP to bind to the nascent transcript and promote termination, our data suggest that the contribution of TRAP-59SL interaction increases the rate of TRAP binding, which, in turn, increases the efficiency of transcription termination.

Molecular basis of TRAP–5′SL RNA interaction in the Bacillus subtilis trp operon transcription attenuation mechanism

“…Previous mutagenesis studies showed that singlestranded nucleotides in the internal loop and the hairpin loop are important for TRAP binding (McGraw et al 2007). To develop a structural model for TRAP-59SL interaction, transcripts containing additional 59SL mutations were characterized.…”

“…Filter binding assays were used to measure apparent dissociation constants (K d ) of TRAP-RNA interactions with mutant transcripts (Table 1). The downstream sequence contained five triplet repeats, as this was previously shown to be the number of repeats in which the 59SL has its greatest influence on TRAP-trp RNA interaction while maintaining moderate affinity (McGraw et al 2007). As previously observed, the G7A and A9G mutations in the internal loop and the A19U and G20A mutations in the hairpin loop decreased the affinity of TRAP-RNA interaction by three-to sixfold (Table 1).…”

“…The length of the upper stem is strongly conserved among organisms containing a trp leader 59SL, particularly those with B. subtilis-like 59SL structures (McGraw et al 2007). To examine the importance of the upper stem length in the B. subtilis 59SL, RNAs containing the 59SL with deletion or insertion of 2 bp in the upper stem ( Fig.…”

“…1) also interacts with TRAP (Sudershana et al 1999;Du et al 2000). The 59SL functions in the attenuation mechanism by increasing the affinity of TRAP for trp leader RNA when the transcript contains nine or fewer repeats, thus increasing the likelihood that TRAP will bind to the nascent transcript in time to promote termination (McGraw et al 2007). Deletion or mutation of the 59SL results in decreased termination efficiency and, therefore, higher expression of the trp operon.…”

“…Deletion or mutation of the 59SL results in decreased termination efficiency and, therefore, higher expression of the trp operon. Nucleotides G7, A8, and A9 from the internal loop, as well as A19 and G20 from the hairpin loop, have been implicated in the mechanism of TRAP-59SL interaction (McGraw et al 2007). These nucleotides are strongly conserved in 59SL structures among organisms closely related to B. subtilis, raising the possibility that the 59SL participates in the trp operon attenuation mechanisms in these organisms as well.…”