A POLR3B-variant reveals a Pol III transcriptome response dependent on La protein/SSB.

RNA polymerase III (Pol III, POLR3) synthesizes tRNAs and other small non-coding RNAs. Human POLR3 pathogenic variants cause a range of developmental disorders, recapitulated in part by mouse models, yet some aspects of POLR3 deficiency have not been explored. We characterized a human POLR3B:c.1625A>G;p.(Asn542Ser) disease variant that was found to cause mis-splicing of POLR3B. Genome-edited POLR3B1625A>G HEK293 cells acquired the mis-splicing with decreases in multiple POLR3 subunits and TFIIIB, although display auto-upregulation of the Pol III termination-reinitiation subunit POLR3E. La protein was increased relative to its abundant pre-tRNA ligands which bind via their U(n)U-3'-termini. Assays for cellular transcription revealed greater deficiencies for tRNA genes bearing terminators comprised of 4Ts than of ≥5Ts. La-knockdown decreased Pol III ncRNA expression unlinked to RNA stability. Consistent with these effects, small-RNAseq showed that POLR3B1625A>G and patient fibroblasts express more tRNA fragments (tRFs) derived from pre-tRNA 3'-trailers (tRF-1) than from mature-tRFs, and higher levels of multiple miRNAs, relative to control cells. The data indicate that decreased levels of Pol III transcripts can lead to functional excess of La protein which reshapes small ncRNA profiles revealing new depth in the Pol III system. Finally, patient cell RNA analysis uncovered a strategy for tRF-1/tRF-3 as POLR3-deficiency biomarkers.

The isolated La-module of LARP1 mediates 3' poly(A) protection and mRNA stabilization, dependent on its intrinsic PAM2 binding to PABPC1.

The protein domain arrangement known as the La-module, comprised of a La motif (LaM) followed by a linker and RNA recognition motif (RRM), is found in seven La-related proteins: LARP1, LARP1B, LARP3 (La protein), LARP4, LARP4B, LARP6, and LARP7 in humans. Several LARPs have been characterized for their distinct activity in a specific aspect of RNA metabolism. The La-modules vary among the LARPs in linker length and RRM subtype. The La-modules of La protein and LARP7 bind and protect nuclear RNAs with UUU-3' tails from degradation by 3' exonucleases. LARP4 is an mRNA poly(A) stabilization factor that binds poly(A) and the cytoplasmic poly(A)-binding protein PABPC1 (also known as PABP). LARP1 exhibits poly(A) length protection and mRNA stabilization similar to LARP4. Here, we show that these LARP1 activities are mediated by its La-module and dependent on a PAM2 motif that binds PABP. The isolated La-module of LARP1 is sufficient for PABP-dependent poly(A) length protection and mRNA stabilization in HEK293 cells. A point mutation in the PAM2 motif in the La-module impairs mRNA stabilization and PABP binding in vivo but does not impair oligo(A) RNA binding by the purified recombinant La-module in vitro. We characterize the unusual PAM2 sequence of LARP1 and show it may differentially affect stable and unstable mRNAs. The unique LARP1 La-module can function as an autonomous factor to confer poly(A) protection and stabilization to heterologous mRNAs.

Signal recognition particle prevents N-terminal processing of bacterial membrane proteins.

Bacterial proteins are synthesized with an N-formylated amino-terminal methionine, and N-formylated peptides elicit innate-immunity responses against bacterial infections. However, the source of these formylated peptides is not clear, as most bacterial proteins are co-translationally deformylated by peptide deformylase. Here we develop a deformylation assay with translating ribosomes as substrates, to show that the binding of the signal recognition particle (SRP) to signal sequences in nascent proteins on the ribosome prevents deformylation, whereas deformylation of nascent proteins without signal sequence is not affected. Deformylation and its inhibition by SRP are not influenced by trigger factor, a chaperone that interacts with nascent chains on the ribosome. We propose that bacterial inner-membrane proteins, in particular those with N-out topology, can retain their N-terminal formyl group during cotranslational membrane insertion and supply formylated peptides during bacterial infections.

A NusG paralogue from Mycobacterium tuberculosis, Rv0639, has evolved to interact with ribosomal protein S10 (Rv0700) but not to function as a transcription elongation-termination factor.

NusG, a well-conserved protein in all the three forms of life, is involved in transcription elongation and termination, as well as in the process of transcription-translation coupling. The existence of species-specific functional, as well as conformational, divergences in NusG makes it an attractive transcription factor to study, especially if it originates from a pathogen. Here, we report functional and conformational characterizations of the Mycobacterium tuberculosis (Mtb) protein Rv0639 that has been annotated as a homologue of Escherichia coli NusG. Rv0639 failed to complement the in vivo functions of E. coli NusG (Ec NusG) and did not exhibit any signature of a transcription elongation-termination factor. However, it retained the ability to bind to its cognate ribosomal protein S10 (Rv0700). Compared with Ec NusG, Rv0639 possesses unique conformational features characterized by altered secondary structures in the C-terminal domain (CTD), an unusually long and disordered linker region between the N-terminal domain (NTD) and CTD, and a folding of its NTD over its CTD. This unusual folded conformation could have imparted specialized functions to this protein, required to adapt the physiology of Mtb. We speculate that in the absence of a bona fide RfaH, a NusG paralogue that is involved in pathogenicity in E. coli, Rv0639 functions as an RfaH-like factor and is involved in pathogenicity using unidentified ops-like sequences in the Mtb genome. And hence, we reannotate Rv0639 as a paralogue of NusG, instead of a homologue.

The role and reliability of rapid bedside diagnostic test in early diagnosis and treatment of bacterial meningitis.

OBJECTIVE: To evaluate the role and reliability of rapid bedside diagnostic test in early diagnosis and treatment of bacterial meningitis in children using reagent strips. METHODS: This prospective, single blinded study was conducted in the Department of Pediatrics of VMMC & Safdarjung Hospital, New Delhi in collaboration with the Department of Microbiology of VMMC & Safdarjung Hospital, New Delhi, over a period of 15 mo (August 2009 to Nov 2010). Seventy-five children aged 3 mo to 12 y admitted in the pediatric ward with suspected diagnosis of acute meningitis were included. All enroled patients underwent lumbar puncture. CSF samples were taken and divided in 2 parts for laboratory evaluation and rapid strip analysis. The sensitivity, specificity, positive predictive value and the negative predictive values of the reagent strips for the diagnosis of bacterial meningitis were calculated. Accuracy of the reagent strips was established using kappa statistics. Latex agglutination for antigen detection and microbiological culture were also done. RESULTS: Highly significant association was observed between CSF examination in routine laboratory method and dipstick method. The number of laboratory values that correlated were- for cells 71(94.63%), for protein 68 (90.67%), for glucose 68(90.67%) out of total 75 cases. The sensitivity and specificity of reagent strip in diagnosing acute bacterial meningitis were 96.7% and 97.8% respectively. The positive predictive and negative predictive values of reagent strip in diagnosing acute bacterial meningitis were 96.7% and 97.8% respectively. Staphylococcus aureus was found to be the most common organism isolated (50%). CONCLUSIONS: Thus reagent strip analysis is a very rapid, reliable and effective method for diagnosis of acute bacterial meningitis in children. Staphylococcus aureus was the most common organism isolated.

A case of periodic hypokalemic paralysis in a patient with celiac disease.

A 4-year-old male child presented with recurrent episodes of diarrhoea for 6-months, each episode associated with weakness of all four limbs and documented hypokalemia who on examination had some pallor, short stature, flaccid quadriparesis with absent DTR. The patient responded clinically and biochemically to potassium supplement. TTG and Intestinal biopsy confirmed celiac disease. Patient was put on gluten free diet and patient is doing well with no recurrence. We present a case of Recurrent hypokalemic paralysis with previously unsuspected celiac disease who was not in celiac crisis.

The moonlighting function of bacteriophage P4 capsid protein, Psu, as a transcription antiterminator.

Psu, a 20-kD bacteriophage P4 capsid decorating protein moonlights as a transcription antiterminator of the Rho-dependent termination. Psu forms specific complex with E.coli Rho protein, and affects the latter's ATP-dependent translocase activity along the nascent RNA. It forms a unique knotted dimer to take a V-shaped structure. The C-terminal helix of Psu makes specific contacts with a disordered region of Rho, encompassing the residues 139-153. An energy minimized structural model of the Rho-Psu complex reveals that the V-shaped Psu dimer forms a lid over the central channel of the Rho hexamer. This configuration of Psu causes a mechanical impediment to the translocase activity of Rho. The knowledge of structural and mechanistic basis of inhibition of Rho action by Psu may help to design peptide inhibitors for the conserved Rho-dependent transcription termination process of bacteria.

Structural and mechanistic basis of anti-termination of Rho-dependent transcription termination by bacteriophage P4 capsid protein Psu.

The conserved bacterial transcription terminator, Rho, is a potent target for bactericidal agents. Psu, a bacteriophage P4 capsid protein, is capable of inducing anti-termination to the Rho-dependent transcription termination. Knowledge of structural and mechanistic basis of this anti-termination is required to design peptide-inhibitor(s) of Rho from Psu. Using suppressor genetics, cross-linking, protein foot-printing and FRET analyses, we describe a conserved disordered structure, encompassing 139-153 amino acids of Rho, as the primary docking site for Psu. Also a neighbouring helical structure, comprising 347-354 amino acids, lining its central channel, plays a supportive role in the Rho-Psu complex formation. Based on the crystal structure of Psu, its conformation in the capsid of the P4 phage, and its interacting regions on Rho, we build an energy-minimized structural model of the Rho:Psu complex. In this model, a V-shaped dimer of Psu interacts with the two diagonally opposite subunits of a hexameric Rho, enabling Psu to form a 'lid' on the central channel of the latter. We show that this configuration of Psu makes the central channel of Rho inaccessible, and it causes a mechanical impediment to its translocase activity.

The first structure of polarity suppression protein, Psu from enterobacteria phage P4, reveals a novel fold and a knotted dimer.

Psu is a capsid decoration protein of bacteriophage P4 and acts as an antiterminator of Rho-dependent transcription termination in bacteria. So far, no structures have been reported for the Psu protein or its homologues. Here, we report the first structure of Psu solved by the Hg(2+) single wavelength anomalous dispersion method, which reveals that Psu exists as a knotted homodimer and is first of its kind in nature. Each monomer of Psu attains a novel fold around a tight coiled-coil motif. CD spectroscopy and the structure of an engineered disulfide-bridged Psu derivative reveal that the protein folds reversibly and reassembles by itself into the knotted dimeric conformation without the requirement of any chaperone. This structure would help to explain the functional properties of the protein and can be used as a template to design a minimal peptide fragment that can be used as a drug against Rho-dependent transcription termination in bacteria.

Crystallization and preliminary X-ray analysis of Psu, an inhibitor of the bacterial transcription terminator Rho.

Psu, a coat protein from bacteriophage P4, inhibits Rho-dependent transcription termination both in vivo and in vitro. The Psu protein is alpha-helical in nature and appeared to be a dimer in solution. It interacts with Rho and affects the ATP binding and RNA-dependent ATPase activity of Rho, which in turn reduces the rate of RNA release from the elongation complex. Crystals of Psu were grown in space group I422 in the presence of PEG, with unit-cell parameters a = b = 148.76, c = 63.38 A and a calculated Matthews coefficient of 2.1 A(3) Da(-1) (41.5% solvent content), assuming the presence of two molecules in the asymmetric unit. A native data set was collected to 2.3 A resolution.

A bacterial transcription terminator with inefficient molecular motor action but with a robust transcription termination function.

Molecular motors such as helicases/translocases are capable of translocating along the single-stranded nucleic acids and unwinding DNA or RNA duplex substrates using the energy derived from their ATPase activity. The bacterial transcription terminator, Rho, is a hexameric helicase and releases RNA from the transcription elongation complexes by an unknown mechanism. It has been proposed, but not directly demonstrated, that kinetic energy obtained from its molecular motor action (helicase/translocase activities) is instrumental in dissociating the transcription elongation complex. Here we report a hexameric Rho analogue (Rv1297, M. tb. Rho) from Mycobacterium tuberculosis having poor RNA-dependent ATP hydrolysis and inefficient DNA-RNA unwinding activities. However, compared to Escherichia coli Rho, it exhibited very robust and earlier transcription termination from the elongation complexes of E. coli RNA polymerase. Bicyclomycin, an inhibitor of ATPase as well as RNA release activities of E. coli Rho, inhibited the ATPase activity of M. tb. Rho with comparable efficiency but was not efficient in inhibiting its transcription termination function. Unlike E. coli Rho, M. tb. Rho was capable of releasing RNA in the presence of nonhydrolyzable analogues of ATP quite efficiently. Also, this termination function most likely does not require NusG, an RNA-release facilitator, as this Rho was incapable of binding to NusG either of M. tb. (Rv0639) or E. coli. These results strongly suggest that the ATPase activity of M. tb. Rho is uncoupled from its transcription termination function and this function may not be dependent on its helicase/translocase activity.

Interaction surface of bacteriophage P4 protein Psu required for complex formation with the transcription terminator Rho.

Rho-dependent transcription termination is an essential function in prokaryotes, and the transcription terminator Rho is highly conserved among different species. The bacteriophage P4 capsid-decoration protein, Psu, interacts specifically with and inhibits the function of Escherichia coli Rho. The interaction surface of Psu involved in interacting with Rho is not known, but knowledge of this is important to understand the mechanism of its action and will be useful to design peptide inhibitor(s) for Rho. We have isolated and characterized seven Psu mutants defective in interacting with Rho and in exerting anti-Rho activity. Conformational probing of Psu revealed that the N-terminal region of the protein folds over onto its central part, forming a globular domain and leaving a solvent-exposed "tail" in the C-terminus. The mutations are located in both of these domains. N-terminal mutants are instrumental in disrupting the N- to C-terminal "cross-talk" in Psu that is required for its structural integrity and its function. Site-specific cross-linking experiments showed that the C-terminal tail preferentially cross-links to Rho and this region of Psu is protected from limited proteolysis when bound to Rho. Therefore, the mutations in this region may have affected the direct interaction of Psu with Rho. We propose that the globular N-terminal domain of Psu confers structural integrity to the functionally important C-terminal tail, which interacts directly with the hexameric Rho.