Characteristics of the essential pathogenicity factor Rv1828, a MerR family transcription regulator from Mycobacterium tuberculosis

Abstract

The gene Rv1828 in Mycobacterium tuberculosis is shown to be essential for the pathogen and encodes for an uncharacterized protein. In this study, we have carried out biochemical and structural characterization of Rv1828 at the molecular level to understand its mechanism of action. The Rv1828 is annotated as helix‐turn‐helix (HTH)‐type MerR family transcription regulator based on its N‐terminal amino acid sequence similarity. The MerR family protein binds to a specific DNA sequence in the spacer region between −35 and −10 elements of a promoter through its N‐terminal domain (NTD) and acts as transcriptional repressor or activator depending on the absence or presence of effector that binds to its C‐terminal domain (CTD). A characteristic feature of MerR family protein is its ability to bind to 19 ± 1 bp DNA sequence in the spacer region between −35 and −10 elements which is otherwise a suboptimal length for transcription initiation by RNA polymerase. Here, we show the Rv1828 through its NTD binds to a specific DNA sequence that exists on its own as well as in other promoter regions. Moreover, the crystal structure of CTD of Rv1828, determined by single‐wavelength anomalous diffraction method, reveals a distinctive dimerization. The biochemical and structural analysis reveals that Rv1828 specifically binds to an everted repeat through its winged‐HTH motif. Taken together, we demonstrate that the Rv1828 encodes for a MerR family transcription regulator.

The conserved actinobacterial transcriptional regulator FtsR controls expression of ftsZ and further target genes and influences growth and cell division in Corynebacterium glutamicum

“…This, together with the strong conservation of our motif among many species, suggests that the motif proposed by us is more likely the correct one. Our results obtained for FtsR and Rv1828 together with the recently published characterization of Rv1828 [40] strongly support the notion that FtsR and its actinobacterial homologs are involved in transcriptional regulation of ftsZ . In line with this, the ftsZ promoter regions of various actinobacterial genera were found to contain DNA sequence motifs similar to the ones determined for FtsR and Rv1828 (Fig.…”

“…Size-exclusion chromatography of affinity-purified FtsR-Strep (calculated mass 28.5 kDa) and comparison to standard proteins indicated that the protein forms a dimer (Additional file 1: Figure S11B). This is in line with the dimeric state of the homologous protein Rv1828 [40]. Purified FtsR-Strep was able to completely shift a 30-bp double-stranded oligonucleotide covering the predicted FtsR-binding site in the ftsZ promoter region at a 2-fold molar excess of the dimeric protein (Fig.…”

“…The binding motif proposed for Rv1828 in a recent study is different but overlaps partially with our motif [40]. Mutational analysis of this binding motif by EMSAs revealed that binding of Rv1828 is not influenced when the specific part of the motif is altered that does not overlap with our proposed binding motif [40]. This, together with the strong conservation of our motif among many species, suggests that the motif proposed by us is more likely the correct one.…”

“…Here the motif was located exactly between the − 10 and − 35 regions of the transcriptional start site P1, which is located at position − 43 relative to the ftsZ translational start [24]. The binding motif proposed for Rv1828 in a recent study is different but overlaps partially with our motif [40]. Mutational analysis of this binding motif by EMSAs revealed that binding of Rv1828 is not influenced when the specific part of the motif is altered that does not overlap with our proposed binding motif [40].…”

“…To test this hypothesis, we expressed ftsR homologs of two pathogenic relatives, CDC7B_1201 of Corynebacterium diphtheriae C7 and Rv1828 of Mycobacterium tuberculosis H37Rv, in the C. glutamicum MB001Δ ftsR strain using pAN6-based expression plasmids, and monitored growth and cell morphology. While this manuscript was in preparation, initial biochemical and structural studies of Rv1828 were published [40], which will be discussed alongside our results. With pAN6-CDC7B_1201 almost full reversal of the growth defect and of the morphological changes was achieved by basal expression without IPTG induction (Additional file 1: Figure S6).…”