cis‐Acting regulatory sequences for antitermination in the transcript of the Bacillus subtilis hut operon and histidine‐dependent binding of HutP to the transcript containing the regulatory sequences

Abstract

SummaryThe location of the cis-acting regulatory region for histidine-dependent antitermination of the Bacillus subtilis hut operon was determined. A secondary structure, whose sequences partially overlap with the downstream terminator, was found in the regulatory region of the hut transcript. Mutational analysis of the regulatory region showed that the secondary structure was required for histidine-dependent antitermination. An electrophoretic mobility-shift assay demonstrated that, in response to the presence of histidine and Mg 21 , purified HutP bound hut RNA bearing putative secondary structure but not RNA lacking the potential to form putative secondary structure. Native gel electrophoresis showed that HutP existed as a hexamer. A filterbinding assay revealed that the concentration of histidine required for half-maximal binding of HutP to RNA was 3.1 mM and that the K d for binding of HutP to RNA was < 0.56 mM in the presence of histidine. These results suggested that putative secondary structure in the regulatory region of hut mRNA could function as an antiterminator to inhibit the formation of the terminator structure and that HutP causes expression of the hut structural genes by binding to the putative antiterminator structure in response to the presence of histidine.

Overall control of nitrogen metabolism in Lactococcus lactis by CodY, and possible models for CodY regulation in Firmicutes

“…It follows that in cases of nutrient limitation leading to a drop in GTP in the cell, CodY repression is alleviated, allowing the expression of many genes involved in cell adaptation to this poorer environmental condition. Many CodY-dependent genes are also regulated by additional mechanisms, allowing their control in response to other environmental factors (Brandenburg et al, 2002;Debarbouille et al, 1999;Ferson et al, 1996;Kim et al, 2003;Mader et al, 2004;Mirel et al, 2000;Nakano et al, 1991;Oda et al, 2000;Ogura et al, 2001;Wray et al, 1997;Yoshida et al, 2003). In B. subtilis, CodY has been shown to directly interact with, in addition to GTP, BCAA, in particular isoleucine (Shivers & Sonenshein, 2004).…”

Studies of the Activation Steps Concurrent to Ligand Binding in δOR and κOR Opioid Receptors Based on Molecular Dynamics Simulations

“…hut mRNA appears to form a stable terminator structure (+498 to 572) in the absence of activated HutP, whereas the region between +489 to +537 seems to form a destabilized RNA structure (ribonucleic anti-terminator, RAT) upon binding to the activated HutP [30]. Our biochemical [30] and recent structural studies [28] revealed that HutP forms a hexamer, as a trimer of dimers. Based on these results, a search was made for repeated sequences within the minimal 79-mer RAT element, and found three UAG repeats that are each separated by a 4-nucleotide spacer region.…”

“…hut mRNA appears to form two alternative secondary structures that depend upon the presence of activated HutP [30]. The regions that fold into alternative structures are located in between +489 to +572 of hut mRNA (Fig.…”

“…Our previous analyses suggested that HutP binds to its cognate RNA only in the presence of L-histidine [28,30]. To asses the ability of HutP to bind to mRNA, reactions were carried out in the presence of L-histidine (10 mM) and Mg 2+ ions (10 mM).…”

Structural insights of HutP-mediated regulation of transcription of the hut operon in Bacillus subtilis

“…Our previous analyses suggested that the HutP protein binds to its cognate RNA only in the presence of L-histidine [28,30]. To analyze the ability of HutP to bind to the mRNA, the reactions are carried out in the presence of L-histidine (10 mM) and Mg 2+ ions (10 mM).…”