Ned Watson

MECHANISM OF RGS4, A GTPASE-ACTIVATING PROTEIN FOR G PROTEIN ALPHA SUBUNITS

GTP hydrolysis by guanine nucleotide-binding proteins, an essential step in many biological processes, is stimulated by GTPase-activating proteins (GAPs). The mechanisms whereby GAPs stimulate GTP hydrolysis are unknown. We have used mutational, biochemical, and structural data to investigate how RGS4, a GAP for heterotrimeric G protein α subunits, stimulates GTP hydrolysis. Many of the residues of RGS4 that interact with Giα1 are important for GAP activity. Furthermore, optimal GAP activity appears to require the additive effects of interactions along the RGS4-Gα interface. GAP-defective RGS4 mutants invariably were defective in binding Gα subunits in their transition state; furthermore, the apparent strengths of GAP and binding defects were correlated. Thus, none of these residues of RGS4, including asparagine 128, the only residue positioned at the active site of Giα1, is required exclusively for catalyzing GTP hydrolysis. These results and structural data (Tesmer, J. G. G., Berman, D. M., Gilman, A. G., and Sprang, S. R. (1997) Cell 89, 251–261) indicate that RGS4 stimulates GTP hydrolysis primarily by stabilizing the transition state conformation of the switch regions of the G protein, favoring the transition state of the reactants. Therefore, although monomeric and heterotrimeric G proteins are related, their GAPs have evolved distinct mechanisms of action.

Expression of GTPase-deficient Giα2 Results in Translocation of Cytoplasmic RGS4 to the Plasma Membrane

The members of a recently identified protein family termed regulators of G-protein signaling (RGS) act as GTPase-activating proteins for certain Gα subunitsin vitro, but their physiological effects in cells are uncertain in the face of similar biochemical activity and overlapping patterns of tissue expression. Consistent with its activity in in vitro GTPase-activating protein assays, RGS4 interacts efficiently with endogenous proteins of the Gi and Gq subclasses of Gα subunits but not with G12α or Gsα. Unlike other RGS proteins such as RGS9, RGS-GAIP, and Sst2p, which have been reported to be largely membrane-associated, a majority of cellular RGS4 is found as a soluble protein in the cytoplasm. However, the expression of a GTPase-deficient Giα subunit (Giα2-Q204L) resulted in the translocation of both wild type RGS4 and a non-Giα-binding mutant (L159F) to the plasma membrane. These data suggest that RGS4 may be recruited to the plasma membrane indirectly by G-protein activation and that multiple RGS proteins within a given cell might be differentially localized to determine a physiologic response to a G-protein-linked stimulus.

Self cleavage of a precursor RNA from bacteriophage T4

We found that a precursor of an RNA molecule from T4-infected Escherichia coli cells (p2Spl; precursor of species 1) has the capacity to cleave itself in a specific position. This cleavage is similar to a cleavage carried out by the aid of a protein, RNase F, that has been previously identified. This cleavage could lead to the maturation of an RNA (species 1) found in T4-infected E. coli cells. The reaction is time and temperature-dependent and is relatively slow as compared to the protein-dependent reaction. It requires at least a monovalent cation and is aided by non-ionic detergents. In the absence of detergent the cleavage can occur but at a reduced rate. The substrate does not contain hidden nicks and a variety of experiments suggest that it does not contain a protein. Moreover, we found no indication that the cleavage is due to contaminating nucleases in the substrate or in the reagents. The intact secondary and tertiary structures of the molecule are necessary for the cleavage to occur. The finding of a self cleaving RNA molecule has interesting evolutionary implications.

Ribonuclease F, a putative processing endoribonuclease from Escherichia coli

Abstract A new endoribonuclease activity, RNase F, was partially purified from Escherichia coli cells. This activity can cleave a precursor RNA molecule (of Species 1), isolated from T4 infected cells, in a specific site. This activity is different from the other three know processing endoribonucleases of E. coli RNase III, RNase E and RNase P.

Consequences of losing ribonuclease III on theEscherichia coli cell

SummaryAn isogenic pair ofEscherichia coli strains, one carrying anrnc+ and the other anrnc− allele (a mutation which reduces the level of ribonuclease III), was compared. Thernc− strain fails to grow at very elevated temperatures (forE. coli) while thernc+ strain does grow exponentially.Assaying the residual RNase III, like activity in extracts of thernc− strain at different pHs and at different temperatures suggested that this residual RNase III like activity is not due to RNase III. This raised the possibility that thernc− strain is devoid of any RNase III activity in the cell. Comparing the decay of newly synthesized RNA and functional decay of β-galactosidase mRNA in such strains revealed that in both strains these parameters proceed in similar rates, which suggests that RNase III is not involved in the metabolism of mRNA. During carbon starvation preexisting total RNA, as well as 23S and 16S rRNA, decay faster in thernc− strain, thus eliminating the possibility that RNase III is the endoribonuclease which initiates the decay of rRNA during starvation (Kaplan and Apirion, 1975a).

Analysis of an Escherichia coli strain carrying physiologically compensating mutations one of which causes an altered ribonuclease III

SummaryA mutant of Escherichia coli, AB105, low in the level of ribonuclease III (an enzyme which degrades double-stranded RNA only), was obtained after treating cells of a ribonuclease I-free strain, A19, with nitrosoguanidine [Kindler, Kiel, and Hofschneider: Molec. gen. Genet. 126, 53–69 (1973)]. By a series of consecutive transductions, using a single E. coli strain, D10, related to the parental strain, as donor, we showed that strain AB105 is separated from strain A19 by at least seven mutations. In order to carry out these transductions, we made use of a number of phenotypic differences which distinguish strains A19 and AB105, such as poor growth on minimal medium or rich medium at any temperature, inability to utilize a variety of carbon sources, etc. After removing some of those mutations it became possible to transduce into such strains the RNase III+ allele from the donor strain D10. Pairs of RNase III+ and RNase III- strains, related one to the other by a single transduction were compared. T4 titers on both types of strains with the same efficiency, while T7 and λ titer better on RNase III+ strains. RNase III+ strains grow slightly better than RNase III- strains at all temperatures, however, at elevated temperatures RNase III+ strains unlike RNase III- strains fail to grow on minimal medium. Thus it seems that the mutation to RNase III- in strain AB105 compensates for some defect that does not permit such strains to grow on minimal medium at elevated temperatures. The enzyme seems to have an indispensible function in the cell but this function is not known yet.

Unaltered stability of newly synthesized RNA in strains of Escherichia coli missing a ribonuclease specific for double-stranded RNA.

SummaryParis of very closely related Escherichia coli strains were prepared, one having the wild-type allele for ribonuclease III, an enzyme which specifically degrades doublestranded RNA, and the other having a mutant RNase III allele. Growth and phage plating efficiency were compared in these strains. The RNase III+ strains grow better than the RNase III- strains and plate T7 and λ phage better, but T4 plates with the same efficiency on both strains.On the other hand, the half lives of newly synthesized RNA as well as of functional β-galactosidase mRNA are similar in both kind of strains. These two parameters, however, are significantly longer in both strains as compared to the original strain from which they were derived. Also, no difference in the differential induction of β-galactosidase was observed between such strains. Thus, we have to conclude that either ribonuclease III does not play a significant role in the functioning and stability of newly synthesized mRNA, or that enough enzymatic activity was left, residual RNase III or some other enzyme to deal with doublestranded regions in the message.

A second gene which affects the RNA processing enzyme ribonuclease P of Escherichia coli

The Escherichia coli RNA processing enzyme RNase P was shown to participate in processing of tRNA [l] and rRNA [2]. Mutations which affect this enzyme were isolated in [3,4]. Recently a mutation affecting RNase P, isolated [3] was mapped near min 82 of the E. coli chromosome [S]. This mutation was designated mpA49. (For nomenclature of mutations affecting RNases see [6].) Here we shall describe the isolation of a second mutation which affects RNase P and show that it maps near min 68 of the E. coli chromosome [7].

Revertants from RNase III negative strains of Escherichia coli.

SummaryE. coli strains carrying the rnc-105 allele do not show any level of RNase III in extracts, grow slower than rnc+ strains at temperatures up to 45°C and fail to grow at 45°C. Revertants which can grow at 45°C were isolated. The vast majority of them still do not grow as fast as rnc+ strains and did not regain RNase III activity. The mutation(s) which caused them are suppressor mutations (physiological suppressors) which do not map in the immediate vicinity of the rnc gene. A few of the revertants regain normal growth, and contain normal levels of RNase III. They do not harbor the rnc-105 allele and therefore are considered to be true revertants. By using purines other than adenine it was possible to isolate rnc+pur- revertants from an rnc-pur- strain with relative ease. They behaved exactly like the true rnc+ revertants isolated from rnc- strains at 45°C.A merodiploid strain which contains the rnc+ gene on an episome behaves exactly like an rnc+ strain with respect to growth and RNA metabolism, eventhough its specific RNase III activity is about 60% of that of an rnc+ strain; thus the level of RNase III is not limiting in the cell.The rnc- strains show a characteristic pattern of transitory molecules, related to rRNA, 30S, 25S, “p23” and 18S, which are not observed in rnc+ strains. This pattern is unchanged in rnc- strains and in the revertants which are still lacking RNase III, regardless of the temperature in which RNA synthesis was examined (30° to 45°C). On the other hand, in the rnc+ strains as well as in the true revertants and the rnc+/rnc- merodiploid, the normal pattern of p16 and p23 is observed at all temperatures. These findings suggest that all the effects observed in RNase III- strains are due to pleiotropic effects of the rnc-105 allele, and that the enzyme RNase III is not essential for the viability of the E. coli cell.

A lethal mutation which affects the maturation of ribosomes

SummaryA temperature sensitive mutant of Escherichia coli which fails to recover from prolonged carbon starvation, was found to be irreversibly killed by exposure to a nonpermissive temperature (43°C), with a half-life of about half an hour. This bacteriocidal effect of the temperature could be reversed by a number of antibiotics which block protein synthesis but not by blocking DNA synthesis. At the nonpermissive temperature, RNA and the protein synthetic capacities decrease before the DNA synthetic capacity is decreased.Analysis of ribosomal proteins and methylation of them did not reveal any consistent differences between the parental and mutant strains. Analysis of the ribosomal RNA revealed that it is being synthesized in similar amounts as in the parental strain at the nonpermissive temperature, however, after chase its level is decreased. Moreover, the 17S precusor RNA is slow to mature to 16S rRNA in the mutant strain at the nonpermissive temperature.Thus, these studies suggest that the mutation studied here affects a late maturation step in the synthesis of the rRNA. Therefore the gene is designated rimH (for ribosomal modification). All the properties bestowed on the mutant strain are caused by a single pleiotropic mutation which maps at min 14 of the E. coli map. Three point transduction crosses suggest the order rimH, leuS, rna, lip. This gene maps outside the two known clusters for ribosomal structural genes.