Latency in Mycobacterium tuberculosis poses a barrier in its complete eradication. Overexpression of certain genes is one of the factors that help these bacilli survive inside the host during latency. Among these genes, rel, which leads to the expression of Rel protein, plays an important role by synthesizing the signaling molecule ppGpp using GDP and ATP as substrates, thereby changing bacterial physiology. In Gram-negative bacteria, the protein is thought to be activated in vivo in the presence of ribosome by sensing uncharged tRNA. In the present report, we show that Rel protein from Mycobacterium smegmatis, which is highly homologous to M. tuberculosis Rel, is functional even in the absence of ribosome and uncharged tRNA. From the experiments presented here, it appears that the activity of Rel Msm is regulated by the domains present at the C terminus, as the deletion of these domains results in higher synthesis activity, with little change in hydrolysis of ppGpp. However, in the presence of tRNA, though the synthesis activity of the full-length protein increases to a certain extent, the hydrolysis activity undergoes drastic reduction. Fulllength Rel undergoes multimerization involving interchain disulfide bonds. The synthesis of pppGpp by the full-length protein is enhanced in the reduced environment in vitro, whereas the hydrolysis activity does not change significantly. Mutations of cysteines to serines result in monomerization with a simultaneous increase in the synthesis activity. Finally, it has been possible to identify the unique cysteine, of six present in Rel, required for tRNA-mediated synthesis of ppGpp.Keywords: Rel protein; stringent response; ppGpp; multimerization; Rel domains Mycobacterium tuberculosis can be categorized as one of the most successful among human pathogens as several decades of research have not yet been able to completely eradicate tuberculosis (TB), the deadly disease caused by this organism. The major barrier toward complete cure from mycobacterial infection is the unique feature, termed ''latency,'' that these bacteria undergo on infection, leading to overexpression of genes that enable the survival of the pathogen within host organisms under oxygen-(Wayne and Hayes 1996) and nutrient-deprived (Nyka 1974) conditions. Such latent bacteria have been known to be confined in calcified lesions, termed ''granulomas,'' which enable the dormant bacteria to resist conventional antibiotics used against active bacilli. It had been proposed that the morphology and hydrophobicity of the in vivo persistors can be mimicked in laboratory cultures by starving bacteria in vitro (Nyka 1974). Under such stress conditions, adaptation to the Reprint requests to: Dipankar Chatterji, Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012, India; e-mail: dipankar@mbu.iisc.ernet.in; fax: +91-80-23600535.Abbreviations: pppGpp, guanosine 39-diphosphate 59-triphosphate; ppGpp, guanosine 39, 59-bis(diphosphate); IPTG, isopropyl b-D-1-thiogalactopyranoside; Ni-NTA, nickel-nitr...

Metabolic pathways contributing to adiposity and aging are activated by the mammalian target of rapamycin complex 1 (mTORC1) and p70 ribosomal protein S6 kinase 1 (S6K1) axis1–3. However, known mTORC1-S6K1 targets do not account for observed loss-of-function phenotypes, suggesting additional downstream effectors4–6. Here we identify glutamyl-prolyl tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes importantly to adiposity and aging. EPRS phosphorylation at Ser999 by mTORC1-S6K1 induces its release from the aminoacyl tRNA multisynthetase complex (MSC), required for execution of noncanonical functions beyond protein synthesis7,8. To investigate physiological function of EPRS phosphorylation, we generated EPRS knock-in mice bearing phospho-deficient Ser999-to-Ala (S999A) and phospho-mimetic (S999D) mutations. Homozygous S999A mice exhibited low body weight, reduced adipose tissue mass, and increased lifespan, thereby displaying notable similarities with S6K1-deficient mice9–11 and mice with adipocyte-specific deficiency of raptor, an mTORC1 constituent12. Substitution of the EPRS S999D allele in S6K1-deficient mice normalized body mass and adiposity, indicating EPRS phosphorylation mediates S6K1-dependent metabolic responses. In adipocytes, insulin stimulated S6K1-dependent EPRS phosphorylation and release from the MSC. Interaction screening revealed phospho-EPRS binds Slc27a1 (i.e., fatty acid transport protein 1, FATP1)13–15, inducing its translocation to the plasma membrane and long-chain fatty acid uptake. Thus, EPRS and FATP1 are terminal mTORC1-S6K1 axis effectors critical for metabolic phenotypes.

Tumors adapt their phenotypes during growth and in response to therapies through dynamic changes in cellular processes. Connexin proteins enable such dynamic changes during development, and their dysregulation leads to disease states. The gap junction communication channels formed by connexins have been reported to exhibit tumor-suppressive functions, including in triple-negative breast cancer (TNBC). However, we find that connexin 26 (Cx26) is elevated in self-renewing cancer stem cells (CSCs) and is necessary and sufficient for their maintenance. Cx26 promotes CSC self-renewal by forming a signaling complex with the pluripotency transcription factor NANOG and focal adhesion kinase (FAK), resulting in NANOG stabilization and FAK activation. This FAK/NANOG-containing complex is not formed in mammary epithelial or luminal breast cancer cells. These findings challenge the paradigm that connexins are tumor suppressors in TNBC and reveal a unique function for Cx26 in regulating the core self-renewal signaling that controls CSC maintenance.

DNA-protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter-RNAP interactions during transcription initiation in the s A -dependent promoters P rrnAPCL1 , P rrnB and P gyr of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation.

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Although sequencing of Mycobacterium tuberculosis genome lead to better understanding of transcription units and gene functions, interactions occurring during transcription initiation between RNA polymerase and promoters is yet to be elucidated. Different stages of transcription initiation include promoter specific binding of RNAP, isomerization, abortive initiation and promoter clearance. We have now analyzed these events with four promoters of M. tuberculosis viz . PgyrB1, PgyrR, PrrnPCL1 and PmetU. The promoters differed from each other in their rates of open complex formation, decay, promoter clearance and abortive transcription. The equilibrium binding and kinetic studies of various steps revealed distinct rate limiting events for each of the promoter, which also differed markedly in their characteristics from the respective promoters of Mycobacterium smegmatis. Surprisingly, the transcription at gyr promoter was enhanced in the presence of initiating nucleotides and decreased in the presence of alarmone, pppGpp, a pattern typically seen with rRNA promoters studied so far. The gyr promoter of M. smegmatis, on the other hand, was not subjected to pppGpp mediated regulation. The marked differences in the transcription initiation pathway seen with rrn and gyr promoters of M. smegmatis and M. tuberculosis suggest that such species specific differences in the regulation of expression of the crucial housekeeping genes could be one of the key determinants contributing to the differences in growth rate and lifestyle of the two organisms. Moreover, the distinct rate limiting steps during transcription initiation of each one of the promoters studied point at variations in their intracellular regulation.

Because of its essential nature, each step of transcription, viz., initiation, elongation, and termination, is subjected to elaborate regulation. A number of transcription factors modulate the rates of transcription at these different steps, and several inhibitors shut down the process. Many modulators, including small molecules and proteinaceous inhibitors, bind the RNA polymerase (RNAP) secondary channel to control transcription. We describe here the first small protein inhibitor of transcription in Mycobacterium tuberculosis. Rv3788 is a homolog of the Gre factors that binds near the secondary channel of RNAP to inhibit transcription. The factor also affected the action of guanosine pentaphosphate (pppGpp) on transcription and abrogated Gre action, indicating its function in the modulation of the catalytic center of RNAP. Although it has a Gre factor-like domain organization with the conserved acidic residues in the N terminus and retains interaction with RNAP, the factor did not show any transcript cleavage stimulatory activity. Unlike Rv3788, another Gre homolog from Mycobacterium smegmatis, MSMEG_6292 did not exhibit transcription-inhibitory activities, hinting at the importance of the former in influencing the lifestyle of M. tuberculosis.T he transcription process of RNA polymerase (RNAP) is controlled by many regulators at different steps (6,11,28). These regulators include both general and operon-specific factors which determine the rate and extent of transcription (2, 3, 28). The functions of these regulators range from the activation of transcription to the repression of the process under different physiological conditions. Apart from transcription factors, different small molecules and antibiotics also target the RNAP to affect transcription. Being the integral component of the essential process, RNAP is a preferred target of a number of antibiotics. The mechanism of action and the binding site for these inhibitors in the multisubunit holoenzyme is distinct. The antibiotics that inhibit RNAP prevent the extension of the nascent RNA beyond the third nucleotide (rifampin and sorangicin) (7,8,31), prevent open complex formation (myxopyronin) (20), or perturb mobile elements in the active center (streptolydigin) (35). The antibacterial peptide microcin J25 inhibits transcription by binding within the RNAP secondary channel and inhibiting nucleoside triphosphate (NTP) uptake (21).A number of structurally similar proteins have been identified from different bacteria which interact with RNAP through the secondary channel. The Gre proteins are the first members of this group of factors to assist RNAP in maintaining transcription accuracy by stimulating the cleavage of aberrant 3= ends of the RNA to resume RNA synthesis (4, 5, 9, 17). Gfh1, DksA, and TraR are structural homologs of Gre factors but do not function like Gre; instead they inhibit the transcription process. Gfh1, which is present in Thermus sp. (13,14,16), inhibits both transcription initiation and elongation (16,33), while DksA of Escherichia c...