Structure and mechanistic features of the prokaryotic minimal RNase P

Endonucleolytic removal of 5'-leader sequences from tRNA precursor transcripts (pre-tRNAs) by RNase P is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein-only versions of the enzyme exert this function in various Eukarya (there termed PRORPs) and in some bacteria (Aquifex aeolicus and close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of Aquifex RNase P (HARPs) are also expressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not represent the main RNase P activity. Here we solved the structure of the bacterial HARP from Halorhodospira halophila by cryo-EM revealing a novel screw-like dodecameric assembly. Biochemical experiments demonstrate that oligomerization is required for RNase P activity of HARPs. We propose that the tRNA substrate binds to an extended spike-helix (SH) domain that protrudes from the screw-like assembly to position the 5'-end in close proximity to the active site of the neighboring dimer. The structure suggests that eukaryotic PRORPs and prokaryotic HARPs recognize the same structural elements of pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for pre-tRNA processing by the prokaryotic HARP system.

Interaction of a photochromic UV sensor protein Rc-PYP with PYP-binding protein

Photoactive yellow protein (PYP) from Halorhodospira halophila is one of typical light sensor proteins. Although its photoreaction has been extensively studied, no downstream partner protein has been identified to date. In this study, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were studied. It was found that UV light-induced a long-lived product (pUV*), which interacts with PBP to form a stable hetero-hexamer (Complex-Ⅱ). The reaction scheme for this interaction was revealed using transient absorption and transient grating methods. Time-resolved diffusion detection showed that a hetero-trimer (Complex-Ⅰ) is formed transiently, which produced Complex-II via a second-order reaction. Any other intermediates, including those from pBL do not interact with PBP. The reaction scheme and kinetics are determined. Interestingly, long-lived Complex-II dissociates upon excitation with blue light. These results demonstrate that Rc-PYP is a photochromic and new type of UV sensor, of which signaling process is similar to that of other light sensor proteins in the visible light region. The photochromic heterogeneous intermolecular interactions formed between PYP and PBP can be used as a novel and useful tool in optogenetics.

Structure and mechanistic features of the prokaryotic minimal RNase P

Endonucleolytic removal of 5'-leader sequences from tRNA precursor transcripts (pre-tRNAs) by RNase P is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein-only versions of the enzyme exert this function in various Eukarya (there termed PRORPs) and in some bacteria (Aquifex aeolicus and close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of Aquifex RNase P (HARPs) are also expressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not represent the main RNase P activity. Here we solved the structure of the bacterial HARP from Halorhodospira halophila by cryo-EM revealing a novel screw-like dodecameric assembly. Biochemical experiments demonstrate that oligomerization is required for RNase P activity of HARPs. We propose that the tRNA substrate binds to an extended spike-helix (SH) domain that protrudes from the screw-like assembly to position the 5'-end in close proximity to the active site of the neighboring dimer subunit. The structure suggests that eukaryotic PRORPs and prokaryotic HARPs recognize the same structural elements of pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for pre-tRNA processing by the prokaryotic HARP system.

Spectroscopic ruler for measuring active-site distortions based on Raman optical activity of a hydrogen out-of-plane vibration

Photoactive yellow protein (PYP), from the phototrophic bacterium Halorhodospira halophila, is a small water-soluble photoreceptor protein and contains p-coumaric acid (pCA) as a chromophore. PYP has been an attractive model for studying the physical chemistry of protein active sites. Here, we explore how Raman optical activity (ROA) can be used to extract quantitative information on distortions of the pCA chromophore at the active site in PYP. We use 13C8-pCA to assign an intense signal at 826 cm−1 in the ROA spectrum of PYP to a hydrogen out-of-plane vibration of the ethylenic moiety of the chromophore. Quantum-chemical calculations based on density functional theory demonstrate that the sign of this ROA band reports the direction of the distortion in the dihedral angle about the ethylenic C=C bond, while its amplitude is proportional to the dihedral angle. These results document the ability of ROA to quantify structural deformations of a cofactor molecule embedded in a protein moiety.

Niche for high abundant extremophilic microbial communities in an ancient crater

Al Wahbah Crater, located in a remote area in western Saudi Arabia as part of The Harrat extinct volcanic chain, is 2 km wide with a depth of 250 m. It is registered by the General Commission for Tourism and National Heritage as an ancient and archaeological site. The crater is subjected to extreme environmental conditions as its bottom is rarely subjected to rainfall and mudflows. Because of high temperature, high evaporation rates and extremely limited rainfall, the crater leaves behind dried thick white sodium phosphate crystals. Here, we studied the chemical composition and the microbial community composition using 16S rRNA pyrosequencing in different vertical layers (2, 20, 40, 60, 80 and 100 cm) of the crater sediment. Total sodium concentrations were 28 000– 46 700 ppm and calcium levels were 31 400– 56 500 ppm. In addition, samples were very sulphuric, with sulphate and sulphite levels exceeding 2157 ppm and 5.54 ppm, respectively. Ferric ions concentrations were <0.2 ppm, while nitrate, ammonium and nitrite levels were <2 ppm, 1.5 ppm and 0.05 ppm, respectively. Archaea dominated the surface and the bottom, while bacteria were most common at 20–60 cm. Extremely halophilic archaea and bacteria includingHalorhabdusspp.Halorubrumspp.,Salinibacter iranicusandHalorhodospira halophilawere identified in all samples. Moreover, the relative abundance ofHalanaerobiaceaeaccounted for 22% of the species in the top of the crater.S. iranicusand species belonging toHalorhabdusandHalorubrumthat were identified between 60 and 100 cm could be considered as extreme organisms.