Publications by authors named "Ram G Nitharwal"

4 Publications

  • Page 1 of 1

A genetically encoded fluorescent tRNA is active in live-cell protein synthesis.

Nucleic Acids Res 2017 04;45(7):4081-4093

Department of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, PA 19107, USA.

Transfer RNAs (tRNAs) perform essential tasks for all living cells. They are major components of the ribosomal machinery for protein synthesis and they also serve in non-ribosomal pathways for regulation and signaling metabolism. We describe the development of a genetically encoded fluorescent tRNA fusion with the potential for imaging in live Escherichia coli cells. This tRNA fusion carries a Spinach aptamer that becomes fluorescent upon binding of a cell-permeable and non-toxic fluorophore. We show that, despite having a structural framework significantly larger than any natural tRNA species, this fusion is a viable probe for monitoring tRNA stability in a cellular quality control mechanism that degrades structurally damaged tRNA. Importantly, this fusion is active in E. coli live-cell protein synthesis allowing peptidyl transfer at a rate sufficient to support cell growth, indicating that it is accommodated by translating ribosomes. Imaging analysis shows that this fusion and ribosomes are both excluded from the nucleoid, indicating that the fusion and ribosomes are in the cytosol together possibly engaged in protein synthesis. This fusion methodology has the potential for developing new tools for live-cell imaging of tRNA with the unique advantage of both stoichiometric labeling and broader application to all cells amenable to genetic engineering.
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http://dx.doi.org/10.1093/nar/gkw1229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5397188PMC
April 2017

Identification and expression of stressosomal proteins in Mycobacterium marinum under various growth and stress conditions.

FEMS Microbiol Lett 2013 May 2;342(2):98-105. Epub 2013 Apr 2.

Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University, SE-751 24 Uppsala, Sweden.

Like other bacteria, Mycobacterium spp. have developed different strategies in response to environmental changes such as nutrient limitations and other different stress situations. We have identified candidate genes (rsb genes) from Mycobacterium marinum involved in the regulation of the activity of the alternative sigma factor, σ(F) . This is a homolog of the master regulator of general stress response, σ(B) , and the sporulation-specific sigma factor, σ(F) , in Bacillus subtilis. The organization of these genes in M. marinum and B. subtilis is similar. Transcriptome and qRT-PCR data show that these genes are indeed expressed in M. marinum and that the levels of expression vary with growth phase and exposure to stress. In particular, cold stress caused a significant rise in the expression of all identified rsb and sigF genes. We discuss these data in relation to what is currently known for other Mycobacterium spp.
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http://dx.doi.org/10.1111/1574-6968.12118DOI Listing
May 2013

DNA binding activity of Helicobacter pylori DnaB helicase: the role of the N-terminal domain in modulating DNA binding activities.

FEBS J 2012 Jan 9;279(2):234-50. Epub 2011 Dec 9.

Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.

Replicative helicases are major motor proteins essential for chromosomal DNA replication in prokaryotes. Usually hexameric in solution, their DNA binding property must have different roles at stages ranging from the loading onto a branched structure at initiation from the origin to the highly processive translocation during elongation. Here, we have analysed the DNA binding activity of Helicobacter pylori (Hp) replicative helicase, DnaB. The results indicate that while the C-terminal region is important for its DNA binding activity, the N-terminus appears to dampen the protein's affinity for DNA. The masking activity of the N-terminus does not require ATP or hexamerization of HpDnaB and can be overcome by deleting the N-terminus. It can also be neutralized by engaging the N-terminus in an interaction with a partner like the C-terminus of DnaG primase. The inhibitory effect of the N-terminus on DNA binding activity is consistent with the 3D homology model of HpDnaB. Electron microscopy of the HpDnaB-ssDNA complex showed that HpDnaB preferentially bound at the ends of linear ssDNA and translocated along the DNA in the presence of ATP. These results provide an insight into the stimulatory and inhibitory effects of different regions of HpDnaB on DNA binding activities that may be central to the loading and translocation functions of DnaB helicases.
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http://dx.doi.org/10.1111/j.1742-4658.2011.08418.xDOI Listing
January 2012

Helicobacter pylori single-stranded DNA binding protein--functional characterization and modulation of H. pylori DnaB helicase activity.

FEBS J 2009 Jan 11;276(2):519-31. Epub 2008 Dec 11.

Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.

Helicobacter pylori, an important bacterial pathogen, causes gastric ulcer and gastric adenocarcinoma in humans. The fundamentals of basic biology such as DNA replication are poorly understood in this pathogen. In the present study, we report the cloning and functional characterization of the single-stranded DNA (ssDNA) binding protein from H. pylori. The N-terminal DNA binding domain shows significant homology with E. coli single-stranded DNA binding protein (SSB), whereas the C-terminal domain shows less homology. The overall DNA-binding activity and tetramerization properties, however, remain unaffected. In in vitro experiments with purified proteins, H. pylori (Hp) SSB bound specifically to ssDNA and modulated the enzymatic ATPase and helicase activity of HpDnaB helicase. HpSSB and HpDnaB proteins were co-localized in sharp, distinct foci in exponentially growing H. pylori cells, whereas both were spread over large areas in its dormant coccoid form, suggesting the absence of active replication forks in the latter. These results confirm the multiple roles of SSB during DNA replication and provide evidence for altered replicative metabolism in the spiral and coccoid forms that may be central to the bacterial physiology and pathogenesis.
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http://dx.doi.org/10.1111/j.1742-4658.2008.06799.xDOI Listing
January 2009
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