A Mutant Rho ATPase from Escherichia Coli That is Temperature-sensitive in the Presence of RNA

Overview

Journal Mol Gen Genet

Specialties Genetics
Molecular Biology

Date 1981 Jan 1

PMID 6165879

Citations 3

Authors

R B Kent

S K Guterman

Affiliations

Soon will be listed here.

Abstract

The Escherichia coli mutant rho-115 suppresses lac operon polarity conferred by the lacZ::IS1 insertion MS319. The ATPase activity of purified rho-115 protein was maximal at 40 degrees C, in contrast to 45 degrees C for rho+. At higher temperatures (50 degrees C, 55 degrees C), the fractions of activities at maximal temperature were consistently lower for rho-115 compared to rho+. The 30-minute time course of rho-115 ATP hydrolysis was linear at 37 degrees C but at 45 degrees C the linear kinetics of hydrolysis reached a plateau between 10 and 15 minutes. The 30-minute time courses for rho+ were linear at both 37 degrees C and 45 degrees C. The rho-115 and rho+ ATPase activities were equally heat-stable during preincubation at 45 degrees C in buffer. Inclusion of ATP during preincubation protected these rho proteins from inactivation to the same extent. The presence of polyC during preincubation protected rho+ activity but produced substantial inactivation of rho-115 ATPase. The presence of polyU during preincubation gave similar results. Concentrations of polyC between 625 ng/ml and 100 micrograms/ml yielded the same extent of rho-115 ATPase inactivation during preincubation at 45 degrees C. Thermal inactivation of rho-115 ATPase by polyC was halted by shifting preincubation temperature from 45 degrees C to 35 degrees C, indicating that polyC-induced destabilization of rho-115 was irreversible.

Citing Articles

Autoregulation of the rho gene of Escherichia coli K-12.

Kung H, Bekesi E, Guterman S, Gray J, Traub L, Calhoun D Mol Gen Genet. 1984; 193(2):210-3.

PMID: 6363877 DOI: 10.1007/BF00330669.

Temperature-sensitive mutant rho-115 rho-RNA binary complexes, and stabilization by substrates and analogues.

Kent R, Guterman S Mol Gen Genet. 1982; 187(2):330-4.

PMID: 6217399 DOI: 10.1007/BF00331139.

Pyrophosphate inhibition of rho ATPase: a mechanism of coupling to RNA polymerase activity.

Kent R, Guterman S Proc Natl Acad Sci U S A. 1982; 79(13):3992-6.

PMID: 6125940 PMC: 346562. DOI: 10.1073/pnas.79.13.3992.

References

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Lowery C, Richardson J . Characterization of the nucleoside triphosphate phosphohydrolase (ATPase) activity of RNA synthesis termination factor p. II. Influence of synthetic RNA homopolymers and random copolymers on the reaction. J Biol Chem. 1977; 252(4):1381-5. View

Shigesada K, Imai M . Studies on the altered rho factor in nitA mutants of Escherichia coli defective in transcription termination. II. Purification and molecular properties of the mutant rho. J Mol Biol. 1978; 120(4):467-86. DOI: 10.1016/0022-2836(78)90349-2. View

Lee F, Yanofsky C . Transcription termination at the trp operon attenuators of Escherichia coli and Salmonella typhimurium: RNA secondary structure and regulation of termination. Proc Natl Acad Sci U S A. 1977; 74(10):4365-9. PMC: 431942. DOI: 10.1073/pnas.74.10.4365. View

Richardson J . An RNA-dependent nucleoside triphosphate phosphohydrolase (ATPase) associated with rho termination factor. Proc Natl Acad Sci U S A. 1974; 71(5):2003-7. PMC: 388373. DOI: 10.1073/pnas.71.5.2003. View

Das A, Merril C, Adhya S . Interaction of RNA polymerase and rho in transcription termination: coupled ATPase. Proc Natl Acad Sci U S A. 1978; 75(10):4828-32. PMC: 336214. DOI: 10.1073/pnas.75.10.4828. View

Kupper H, Sekiya T, Rosenberg M, Egan J, Landy A . A rho-dependent termination site in the gene coding for tyrosine tRNA su3 of Escherichia coli. Nature. 1978; 272(5652):423-8. PMC: 1994828. DOI: 10.1038/272423a0. View

Jovin T, Chrambach A, Naughton M . AN APPARATUS FOR PREPARATIVE TEMPERATURE-REGULATED POLYACRYLAMIDE GEL ELECTROPHORESIS. Anal Biochem. 1964; 9:351-69. DOI: 10.1016/0003-2697(64)90192-7. View

Ratner D . Evidence that mutations in the suA polarity suppressing gene directly affect termination factor rho. Nature. 1976; 259(5539):151-3. DOI: 10.1038/259151a0. View

10.

MALAMY M . Frameshift mutations in the lactose operon of E. coli. Cold Spring Harb Symp Quant Biol. 1966; 31:189-201. DOI: 10.1101/sqb.1966.031.01.027. View

11.

Fuller R, Platt T . The attenuator of the tryptophan operon in E.coli: rho-mediated release of RNA polymerase from a transcription termination complex in vitro. Nucleic Acids Res. 1978; 5(12):4613-23. PMC: 342776. View

12.

Howard B, de Crombrugghe B . ATPase activity required for termination of transcription by the Escherichia coli protein factor rho. J Biol Chem. 1976; 251(8):2520-4. View

13.

Guterman S, Howitt C . Rifampicin supersensitivity of rho strains of E. coli, and suppression by sur mutation. Mol Gen Genet. 1979; 169(1):27-34. DOI: 10.1007/BF00267541. View

14.

Richardson J, Grimley C, Lowery C . Transcription termination factor rho activity is altered in Escherichia coli with suA gene mutations. Proc Natl Acad Sci U S A. 1975; 72(5):1725-8. PMC: 432618. DOI: 10.1073/pnas.72.5.1725. View

15.

Darlix J . The functions of rho in T7-DNA transcription in vitro. Eur J Biochem. 1973; 35(3):517-26. DOI: 10.1111/j.1432-1033.1973.tb02868.x. View

16.

Minkley Jr E . Letter: functional form of RNA synthesis termination factor rho. J Mol Biol. 1973; 78(3):577-80. DOI: 10.1016/0022-2836(73)90479-8. View

17.

Guarente L, Beckwith J . Mutant RNA polymerase of Escherichia coli terminates transcription in strains making defective rho factor. Proc Natl Acad Sci U S A. 1978; 75(1):294-7. PMC: 411233. DOI: 10.1073/pnas.75.1.294. View

18.

Galluppi G, Richardson J . ATP-induced changes in the binding of RNA synthesis termination protein Rho to RNA. J Mol Biol. 1980; 138(3):513-39. DOI: 10.1016/s0022-2836(80)80016-7. View

19.

Rosenberg M, Court D, Shimatake H, Brady C, WULFF D . The relationship between function and DNA sequence in an intercistronic regulatory region in phage lambda. Nature. 1978; 272(5652):414-23. DOI: 10.1038/272414a0. View

20.

Wu A, Chapman A, Platt T, Guarente L, Beckwith J . Deletions of distal sequence after termination of transcription at the end of the tryptophan operon in E. coli. Cell. 1980; 19(4):829-36. DOI: 10.1016/0092-8674(80)90073-2. View