Pressure pushes tRNALys3 into excited conformational states.

Conformational dynamics play essential roles in RNA function. However, detailed structural characterization of excited states of RNA remains challenging. Here, we apply high hydrostatic pressure (HP) to populate excited conformational states of tRNALys3, and structurally characterize them using a combination of HP 2D-NMR, HP-SAXS (HP-small-angle X-ray scattering), and computational modeling. HP-NMR revealed that pressure disrupts the interactions of the imino protons of the uridine and guanosine U-A and G-C base pairs of tRNALys3. HP-SAXS profiles showed a change in shape, but no change in overall extension of the transfer RNA (tRNA) at HP. Configurations extracted from computational ensemble modeling of HP-SAXS profiles were consistent with the NMR results, exhibiting significant disruptions to the acceptor stem, the anticodon stem, and the D-stem regions at HP. We propose that initiation of reverse transcription of HIV RNA could make use of one or more of these excited states.

Iron uptake and transport by the carboxymycobactin-mycobactin siderophore machinery of Mycobacterium tuberculosis is dependent on the iron-regulated protein HupB.

Iron-starved Mycobacterium tuberculosis utilises the carboxymycobactin-mycobactin siderophore machinery to acquire iron. These two siderophores have high affinity for ferric iron and can withdraw the metal ion from insoluble iron hydroxides and iron-binding proteins. We first reported HupB, a multi-functional mycobacterial protein to be associated with iron acquisition in M. tuberculosis. This 28 kDa cell wall protein, up regulated upon iron limitation functions as a transcriptional activator of mycobactin biosynthesis and is essential for the pathogen to survive inside macrophages. The focus of this study is to understand the role of HupB in iron uptake and transport by the carboxmycobactin-mycobactin siderophore machinery in M. tuberculosis. Experimental approaches included radiolabelled iron uptake studies by viable organisms and protein-ligand binding studies using the purified HupB and the two siderophores. Uptake of 55Fe-carboxymycobactin by wild type M. tuberculosis (WT M.tb.H37Rv) and not by the hupB KO mutant (M.tb.ΔhupB) showed that HupB is necessary for the uptake of ferri-carboxymycobactin. Additionally, the radiolabel recovery was high in HupB-incorporated liposomes upon addition of the labelled siderophore. Bioinformatic and experimental studies using spectrofluorimetry, CD analysis and surface plasmon resonance not only confirmed the binding of HupB with ferri-carboxymycobactin and ferri-mycobactin but also with free iron. In conclusion, HupB is established as a ferri- carboxymycobactin receptor and by virtue of its property to bind ferric iron, functions as a transporter of the ferric iron from the extracellular siderophore to mycobactin within the cell envelope.

A putative merR family transcription factor Slr0701 regulates mercury inducible expression of MerA in the cyanobacterium Synechocystis sp. PCC6803.

In cyanobacteria, genes conferring mercury resistance are not organized as mer-operon, unlike in other bacterial phyla. Synechocystis contains only a putative MerR regulator, Slr0701, and a mercury reductase, MerA, located aside from each other in the genome. The slr0701-mutant showed reduction in photosynthetic activity and reduced tolerance to mercury compared to the wild-type. The incubation of wild-type cells with HgCl2 resulted in the upregulation of slr0701 and slr1849 genes whereas mercury-induced expression was not observed in the slr0701-mutant. Slr0701 binds to a conserved cis-regulatory element located in the upstream of slr1849 and slr0701 ORFs. The same element was also identified in the upstream of other cyanobacterial homologs. Slr0701 binds to cis-regulatory element with faster association and slower dissociation rates in the presence of HgCl2 . Although these genes were constitutively expressed, the addition of HgCl2 enhanced their promoter activity suggesting that mercury-bound Slr0701 triggers induced expression of these genes. The enhanced promoter activity could be attributed to the observed secondary structural changes in Slr0701 in the presence of HgCl2 . For the first time, we demonstrated the mechanism of merA regulation in a cyanobacterium, Synechocystis. Although merA and merR genes are distantly located on the cyanobacterial genome and distinct from other bacterial mer-operons, the transcriptional regulatory mechanism is conserved.