Hut mRNA Research Articles

Metal ion-dependent anti-termination of transcriptional regulation of ribonucleoprotein complexes.

Anti-terminator proteins are frequently used by bacteria to sense a specific metabolite signal and direct RNA polymerase to either terminate or continue transcription of the genes downstream of an operon. One such protein is HutP, which binds to upstream cis-regulatory sequences to regulate expression of the histidine utilization (hut) operon in Bacillus subtilis. HutP must be activated by L-histidine and divalent metal ions before binding to hut mRNA; binding of activated HutP prevents termination of transcription. Thus, HutP appears to regulate the hut operon in a unique fashion in this class of regulatory proteins. To understand gene (hut operon) regulation by HutP, we performed several biochemical and structural studies. These studies reveal events in the regulatory mechanism, starting with the activation of HutP and ending with the unwinding of hut terminator RNA. In this review, we describe the unique regulatory mechanisms commonly used by many Bacillus species.

Crystal structure of the single-stranded RNA binding protein HutP from Geobacillus thermodenitrificans

RNA binding proteins control gene expression by the attenuation/antitermination mechanism. HutP is an RNA binding antitermination protein. It regulates the expression of hut operon when it binds with RNA by modulating the secondary structure of single-stranded hut mRNA. HutP necessitates the presence of l-histidine and divalent metal ion to bind with RNA. Herein, we report the crystal structures of ternary complex (HutP–l-histidine–Mg2+) and EDTA (0.5M) treated ternary complex (HutP–l-histidine–Mg2+), solved at 1.9Å and 2.5Å resolutions, respectively, from Geobacillus thermodenitrificans. The addition of 0.5M EDTA does not affect the overall metal-ion mediated ternary complex structure and however, the metal ions at the non-specific binding sites are chelated, as evidenced from the results of structural features.

Alternative binding modes of l-histidine guided by metal ions for the activation of the antiterminator protein HutP of Bacillus subtilis

Anti-terminator proteins control gene expression by recognizing control signals within cognate transcripts and then preventing transcription termination. HutP is such a regulatory protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences in hut mRNAs. During the anti-termination process, l-histidine and a divalent ion are required for hutP to bind to the specific sequence within the hut mRNA. Our previous crystal structure of the HutP-l-histidine-Mg(2+)-RNA ternary complex demonstrated that the l-histidine ligand and Mg(2+) bind together such that the backbone nitrogen and carboxyl oxygen of l-histidine coordinate with Mg(2+). In addition to the Mg(2+), other divalent ions are also known to efficiently support the l-histidine-dependent anti-termination of the hut operon, and the best divalent ion is Zn(2+). In this study, we determined the crystal structure of the HutP-l-histidine-Zn(2+) complex and found that the orientation of l-histidine coordinated to Zn(2+) is reversed relative to that of l-histidine coordinated to Mg(2+), i.e., the imidazole side chain nitrogen of l-histidine coordinates to Zn(2+). This alternative binding mode of the l-histidine ligand to a divalent ion provides further insight into the mechanisms responsible for the activation of RNA binding during the hut anti-termination process.

Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis

HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg2+ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn2+, Zn2+ and Cd2+, followed by Mg2+, Co2+ and Ni2+, while Cu2+, Yb2+ and Hg2+ were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg2+, Mn2+ and Ba2+), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition.

Crystallization and preliminary X-ray diffraction studies of the metal-ion-mediated ternary complex of the HutP protein with l-histidine and its cognate RNA

HutP is an RNA-binding protein that regulates the expression of the Bacillus subtilis hut operon by binding to cis-acting regulatory sequences within hut mRNA, exclusively in the presence of l-histidine. We recently solved the crystal structure of a binary complex (HutP with an l-histidine analog) that revealed a novel RNA-binding fold, and identified the important residues that interact with the l-histidine analog. In addition, we have defined the minimal RNA binding segment that is required for HutP recognition. Interestingly, we showed that ternary complex formation depends on the availability of not only l-histidine but also divalent metal ions. Here we report the crystallization and preliminary X-ray diffraction analysis of the HutP ternary complex. The ternary complex was crystallized in the presence of Mg 2+ along with l-histidine and hut mRNA, using the hanging drop vapor diffusion method. The crystal belongs to the R3 space group, with unit cell parameters a= b=75.30 Å, c=133.8 Å. A complete data set at 1.60 Å was collected.

Identification of important chemical groups of the hut mRNA for HutP interactions that regulate the hut operon in Bacillus subtilis.

HutP is an RNA binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus species by binding to cis-acting regulatory sequences on hut mRNA. We recently solved the HutP crystal structure, which revealed a novel fold where three dimers are arranged in a 3-fold axis to form the hexamer. We also identified a minimal RNA binding element sufficient for HutP binding: three UAG trinucleotide motifs, each separated by 4 nt, located just upstream of the terminator. In the present study we have identified important RNA chemical groups essential for HutP interactions, by combining an in vitro selection strategy and analyses by site-specific base substitutions. These analyses suggest that each HutP molecule recognizes one UAG motif, where the first base (U) can be substituted with other bases, while the second and third bases (A and G) are required for the interactions. Further analyses of the chemical groups of the A and G bases in the UAG motif by modified base analogs suggested the importance of the exocyclic NH2 group in these bases. Also, in this motif, only the 2'-OH group of A is important for HutP recognition. Considering the important chemical groups identified here, as well as the electrostatic potential analysis of HutP, we propose that Glu137 is one of the important residues for the HutP-RNA interactions.

Crystal structure of activated HutP; an RNA binding protein that regulates transcription of the hut operon in Bacillus subtilis.

HutP is an L-histidine-activated RNA binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on the hut mRNA. The crystal structure of HutP complexed with an L-histidine analog showed a novel fold; there are four antiparallel beta strands in the central region of each monomer, with two alpha helices each on the front and back. Two HutP monomers form a dimer, and three dimers are arranged in crystallographic 3-fold symmetry to form a hexamer. A histidine analog was located in between the two monomers of HutP, with the imidazole group of L-histidine hydrogen bonded to Glu81. An activation mechanism is proposed based on the identification of key residues of HutP. The HutP binding region in hut mRNA was defined: it consists of three UAG trinucleotide motifs separated by four spacer nucleotides. Residues of HutP potentially important for RNA binding were identified.

Analysis of HutP-dependent transcription antitermination in the Bacillus subtilis hut operon: identification of HutP binding sites on hut antiterminator RNA and the involvement of the N-terminus of HutP in binding of HutP to the antiterminator RNA.

We investigated HutP-dependent transcription antitermination of the Bacillus subtilis hut operon. In vitro transcription assays with the B. subtilissigmaA-containing RNA polymerase indicated that HutP inhibits transcription termination at the internal terminator by binding to the antiterminator on hut mRNA in the presence of histidine. Ethylnitrosourea modification interference assays and mutational analyses of the interference sites showed that interaction of HutP with a region containing three UAG trinucleotide sequences, which is located on top of the antiterminator structure, is critical for hut antitermination in vivo. Results from kinetic analysis of binding of HutP to RNA containing various portions of the antiterminator sequences indicated that secondary structure is required for binding of HutP to the region containing three UAG triplets in the antiterminator. The in vivo HutP antiterminator activity was reduced by the mutations in the N-terminal region of HutP. The HutP variants with H4A, R7A, I9A and Q26A mutations exhibited reduced binding affinities to the antiterminator RNA in vitro. A 25-mer peptide consisting of amino acid residues 2-26 of HutP bound to the antiterminator RNA. These results indicated that the N-terminus of HutP is involved in binding of HutP to the antiterminator RNA.

Allosteric activation of HutP protein, that regulates transcription of hut operon in Bacillus subtilis, mediated by various analogs of histidine

HutP is an RNA-binding protein which regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on the hut mRNA in the presence of L-histidine. In the case of HutP-RNA interactions, L-histidine plays an allosteric activation role i.e. only the activated HutP specifically recognize the hut mRNA. In the present study, we analyzed various analogs of L-histidine to evaluate the important functional groups of L-histidine that is responsible for the activation of HutP. Our analysis clearly suggests that imidazole group of a L-histidine plays a vital role for the activation. Our analysis also revealed efficient analogs of L-histidine for the activation, for example, L-histidine beta-naphthylamide and L-Histidine benzyl ester.

Crystallization and preliminary X-ray diffraction studies of HutP protein: an RNA-binding protein that regulates the transcription of hut operon in Bacillus subtilis.

HutP is an RNA-binding protein and regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on hut mRNA. A single point mutant (Val51Ile), which has increased affinity for the regulatory sequences, were purified and crystallized by the hanging drop vapor diffusion method. The space group was P2(1)3 with unit cell dimension a = b = c = 95.60 A resolution, and contain two molecules in the asymmetric unit. A complete MAD data set of 3.0 A resolution were collected at the LIII edge of Hg.

Crystallization and preliminary X-ray diffraction studies of HutP protein: an RNA-binding protein that regulates the transcription of hut operon in Bacillus subtilis

HutP is an RNA-binding protein and regulates the expression of the histidine utilization ( hut) operon in Bacillus subtilis by binding to cis-acting regulatory sequences on hut mRNA. HutP and its mutant, which has increased affinity for the regulatory sequences, were purified and crystallized by the hanging-drop vapor diffusion method. The space group was P2 13 with unit cell dimensions a=b=c=95.6 A ̊ for HutP and a=b=c=96.8 A ̊ for the mutant. Complete data sets of 3.0-Å resolution for wild-type HutP and of 2.70-Å resolution for the mutant HutP were collected.

Analysis of histidine-dependent antitermination in Bacillus subtilis hut operon

We have previously shown that a positive regulator, HutP, of Bacillus subtilis hut operon is a RNA binding protein. Here, we report precise binding site of HutP in cis-regulatory region on hut mRNA and the role of HutP in histidine-dependent antitermination of hut expression. Ethylnitrosourea modification interference assay showed that four binding sites of HutP were found in the cis-regulatory sequences and were located at the stem and the internal loop of an antiterminator structure. In vitro transcription assay indicated that HutP suppressed transcription termination in the presence of histidine. These results suggest that HutP function as an antiterminator in response to the presence of histidine.

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

The 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 Mg2+, 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 filter-binding assay revealed that the concentration of histidine required for half-maximal binding of HutP to RNA was 3.1 mM and that the Kd for binding of HutP to RNA was approximately 0.56 microM 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.