| 1. |
- Masuda, Isao, et al.
(författare)
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A genetically encoded fluorescent tRNA is active in live-cell protein synthesis
- 2017
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Ingår i: Nucleic Acids Research. - : OXFORD UNIV PRESS. - 0305-1048 .- 1362-4962. ; 45:7, s. 4081-4093
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Tidskriftsartikel (refereegranskat)abstract
- 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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| 2. |
- Nitharwal, Ram G., et al.
(författare)
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DNA binding activity of Helicobacter pylori DnaB helicase : the role of the N-terminal domain in modulating DNA binding activities
- 2012
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 279:2, s. 234-250
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Tidskriftsartikel (refereegranskat)abstract
- 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 proteins 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 HpDnaBssDNA 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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| 3. |
- Sharma, Atul, et al.
(författare)
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Helicobacter pylori single-stranded DNA binding protein - functional characterization and modulation of H-pylori DnaB helicase activity
- 2009
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 276:2, s. 519-531
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Tidskriftsartikel (refereegranskat)abstract
- 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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| 4. |
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