Degradation of Guanosine 3'-diphosphate 5'-diphosphate in vitro by the spoT Gene Product of Escherichia coli

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

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In vitro functional characterization of overproduced Escherichia coli katF/rpoS gene product

Biochemistry ◽

10.1021/bi00092a021 ◽

1993 ◽

Vol 32 (41) ◽

pp. 11112-11117 ◽

Cited By ~ 48

Author(s):

Lam H. Nguyen ◽

Debra B. Jensen ◽

Nancy E. Thompson ◽

Daniel R. Gentry ◽

Richard R. Burgess

Keyword(s):

Escherichia Coli ◽

Gene Product ◽

Functional Characterization ◽

Rpos Gene

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Studies on In Vitro DNA Synthesis Purification of dna C Gene Product Containing dna D Activity from Escherichia coli

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.70.8.2369 ◽

1973 ◽

Vol 70 (8) ◽

pp. 2369-2373 ◽

Cited By ~ 18

Author(s):

S. Wickner ◽

I. Berkower ◽

M. Wright ◽

J. Hurwitz

Keyword(s):

Escherichia Coli ◽

Dna Synthesis ◽

Gene Product

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The relationship between the spoT gene, the synthesis of stable RNA, ribosomal proteins, and the ββ′ subunits of RNA polymerase following a nutritional shiftup of Escherichia coli

Canadian Journal of Biochemistry ◽

10.1139/o78-081 ◽

1978 ◽

Vol 56 (6) ◽

pp. 528-533 ◽

Cited By ~ 6

Author(s):

Stephen M. Boyle ◽

Frederick Chu ◽

Nathan Brot ◽

Bruce H. Sells

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Rna Synthesis ◽

Transient Increase ◽

Spot Gene ◽

The Relationship

The level of ppGpp and rates of synthesis of stable RNA, ribosomal protein, and the β and β′ subunits of RNA polymerase were measured following a nutritional shiftup in Escherichia coli strains, NF 929 (spoT+) and NF 930 (spoT'−). In the spoT+ strain, ppGpp levels decreased 50% within 2 min following shiftup, and the rates of synthesis of stable RNA, ribosomal proteins, and the β and β′ subunits of RNA polymerase increased with little or no lag. In contrast, in the spoT− strain, ppGpp levels transiently increased 40% during the first 6 min following shiftup. An inhibition in the rate of stable RNA synthesis and a delay in the increased synthesis of ribosomal proteins and β and β′ subunits occurred concurrently with the transient increase in ppGpp. In addition, the DNA-dependent synthesis in vitro of the β and β′ subunits of RNA polymerase was inhibited by physiological levels of ppGpp. Because of the timing and magnitude of the changes in ppGpp levels in the spoT− strain versus the timing when the new rates of stable RNA, ribosomal protein, and β and β′ subunits synthesis are reached, it is concluded that ppGpp is not the sole element regulating the expression of these genes.

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