Gene affecting longevity of messenger RNA: a mutant of Escherichia coli with altered mRNA stability.

Summary we have screened 897 temperature sensitive growth mutants ofE. coli for mutant strains showing longer mRNA half-life. The fate of pulse-labelled RNA was examined at 42° C after cessation of RNA synthesis and with prior exposure to nonpermissive temperature (42° C). Eight stains showed altered turn-over of RNA (presumably mRNA), and further analysis on mutant strain JE15144 indicated that the stability of pulse-labeled RNA as well as of tryptophan (trp) mRNA increased four to seven fold over its parental strain at 42° C. At 4 min or 10 min after addition of rifampicin, some 70 to 80% of polyribosome in the growing cells could still be conserved in JE15144 cultured at the nonpermissive temperature while little, if any, polyribosomes remained in its parental strain (PA3092) under the same condition. Two generation times were required for complete stoppage of growth of this mutant strain after shifting to 42° C, and protein synthesis continued at a significant, but slightly reduced, rate at 42° C. However, functional decay of mRNA in the mutant strain, with respect to the capacity for producing peptides, appeared to be similar to the parent strain, with half-lives of 3.5 min in PA3092 and 4.7 min in JE15144.     

Differential modes of chemical decay for early and late lambda messenger RNA.

In the presence of rifampicin, chemical decay of pulse-labeled phage λ “early” messenger RNA occurred exponentially, with a half-life of 2.2 minutes at 42 °C, compared to 1.3 minutes for the host mRNA. In contrast, mRNA synthesized late during λ development exhibited a characteristic complex decay curve not previously observed for microbial mRNA; initial slow decay of about 25% during 4 to 4.5 minutes was followed by rapid exponential decay of the remainder with a half-life of 1.5 minutes. Sucrose density-gradient analyses of the RNA revealed that during the phase of slow decay, the λ-specific mRNA was progressively fragmented.     

Stability of rapidly labelled messenger ribonucleic acid in Bacillus amyloliquefaciens during the phases of minimum and maximum extracellular enzyme formation.

The stability of rapidly labelled hybridizable messenger RNA in both exponential and post-exponential phase cells of Bacillus amyloliquefaciens was measured in terms of the rate of loss of its radioactivity. In the exponential phase, where 96% of the mRNA was specific for cell proteins and only 4% was exoprotein mRNA, the label was lost exponentially from the rapidly labelled hybridizable mRNA fraction with a half-life of six minutes at 30 °C. The antibiotic rifampicin, at a concentration of 10 μg/ml, had no effect on the characteristics of decay of this exponential-phase mRNA. In the post-exponential phase, where there were equal amounts of cell protein and exoprotein-specific mRNA, rapidly labelled hybridizable mRNA decayed exponentially in the presence of rifampicin (10 μg/ml), with a half-life of six minutes at 30 °C. In the absence of rifampicin the characteristics of decay were more complex. The evidence available suggested that this was due to the superimposition of a component attributable to reincorporation of degradation products of radioactive RNA on the characteristic exponential decay pattern of the post-exponential mRNA.Measurement of the stability of active mRNA, by studying the loss of ability to incorporate l-[¹⁴C]leucine into protein in the presence of rifampicin (10 μg/ml), gave half-lives of 4.5 minutes and six minutes, respectively, for exponential and post-exponential material.     

A mutant of Escherichia coli blocked in peptide elongation: altered elongation factor Ts

Among our transfer RNA-dependent growth mutants, one, HAK88, was found that carries an altered elongation factor Ts. The activity of mutant EFTs to bind GDP to EFTu, or to form the ternary complex (aminoacyl-tRNA-EFTu-GTP) is thermolabile. The effect of magnesium on the formation of EFTu-GDP from the EFTu-EFTs complex of HAK8 shows that a four to fivefold increase of the duplex formation occurs when the magnesium concentration is increased from 10^?6m to 10^?2m at 0 °C and at 41 °C. However, at higher temperatures, formation of the binary EFTu-GDP from the EFTu-EFTs complex of HAK88 is depressed, even at 10^?3m to 10^?2m-magnesium. The binding of GDP to the wild-type or mutant EFTu-EFTs complex at 0 °C and 42 °C indicates that the formation of EFTu-GDP is inhibited at 42 °C only when mutant complex is used for the assay. Binding of GTP to complete bacteriophage Qβ replicase (which is known to contain EFTs) formed in phage-infected HAK88 is also inhibited at 42 °C.     

Function of bacteriophage Qbeta replicase containing an altered subunit IV

In order to elucidate the function of elongation factor Ts in Qβ replicase, enzyme was obtained from a Qβ-infected Escherichia coli mutant HAK88, which carries an altered EFTs² with a thermolabile catalytic activity. HAK88 Qβ replicase was found to be quite unstable at 42 °C. Further studies indicated that the mutant enzyme exhibits temperature sensitivity with regard to GTP binding ability but not with Qβ RNA and poly(C) binding. These results suggest that the function of EFTs in Qβ replicase is closely related to the binding of GTP to the enzyme.A defect in Qβ replicase also appears when it is reconstituted from the Qβ replicase subunit complex I–II and the HAK88 EFTu-EFTs complex. Several lines of evidence obtained by using the reconstituted enzyme suggest strongly that the EFTs function is involved specifically in initiation of RNA synthesis, but not in the elongation reaction.     

Conditional rifampicin sensitivity of arif mutant ofEscherichia coli: rifampicin induced changes in transcription specificity

Arif mutantof Escherichia coli that exhibits medium and temperature-dependent sensitivity to rifampicin is described. In the absence of rifampicin, this strain grows in minimal and rich media at 30°C and 42°C. In its presence it is viable in rich medium at both temperatures, but in minimal medium only at 30°C. In minimal-rifampicin medium at the higher temperature, RNA synthesis is decreased. The addition of certain divalent salts (MgSO₄, CaCl₂, BaCl₂) in excess, or chelators (EDTA, EGTA, o-phenanthrolein) greatly increase viability in minimal-rifampicin medium at 42°C. Excess MgSO₄ (10 mM) also increases the rate of RNA synthesis in the same medium. A model is proposed wherein therif mutation is suggested to cause a structural change in RNA polymerase that allows the binding of rifampicin and other ligands at 42°C. Rifampicin-binding is suggested to alter the conformation of RNA polymerase, impairing its ability to express genes required for growth in minimal medium. Implicit in this view is the assumption that these genes are structurally different from those expressed in rich medium in respect of certain template features recognized by RNA polymerase.     

Early to late switch in bacteriophage T7 development: functional decay of T7 early messenger RNA

Infection of ultraviolet light-irradiated Escherichia coli with T7 phage in the presence of chloramphenicol results in synthesis of T7 early messenger RNA but not late mRNA. T7 early mRNA accumulates in terms of acid-insoluble, T7 DNA-hybridizable RNA. However, messenger activity of the same RNA decays rapidly with a half-life of about 6.5 minutes at 30 °C when tested for the ability to direct in vitro protein synthesis. This functional decay of T7 early mRNA is attributable to a loss of structural integrity of the RNA. Polyacrylamide-agarose gel electrophoresis shows that T7 early mRNAs are cleaved, generating smaller-size RNAs. Kinetics of the appearance of T7-specific RNA polymerase, one of the early gene products, during normal T7 infection show that the capacity of the cells to produce the enzyme decays very rapidly when early mRNA synthesis is terminated either by rifampicin or by a natural mechanism programmed by T7. Preferential synthesis of late proteins in the presence of chemically stable early mRNA late in T7 infection may be explained by the observed functional decay of early mRNA.     

Characterization of Mutants of Escherichia coli Temperature-Sensitive for Ribonucleic Acid Regulation: an Unusual Phenotype Associated with a Phenylalanyl Transfer Ribonucleic Acid Synthetase Mutant

A mutant strain AA-522, temperature-sensitive for protein synthesis, was isolated from a stringent strain (CP-78) of Escherichia coli K-12. The mutant strain has a relaxed phenotype at the nonpermissive growth temperature. Protein synthesis stops completely at 42 C, whereas the rate of ribonucleic acid (RNA) synthesis is maintained at 20% of the 30 C rate. Sucrose-gradient centrifugation analysis of RNA-containing particles formed at 42 C indicated the presence of “relaxed particles.” These particles possess 16S and 23S RNA and are precursors to normal 50S and 30S ribosomal subunits. A search for the temperature-sensitive protein responsible for the halt in protein synthesis implicated phenylalanyl transfer RNA (tRNA) synthetase. Essentially no enzyme activity is detected in vitro at 30 or 40 C. Analysis of phenylalanyl tRNA synthetase activity in revertants of strain AA-522 indicated the presence of intragenic suppressor mutations. Revertants of strain AA-522 analyzed for the relaxed response at 42 C were all stringent; strain AA-522 was stringent at 30 C. These data indicate that a single mutation in phenylalanyl tRNA synthetase is responsible for both a block in protein synthesis and the relaxed phenotype at 42 C.     

An Escherichia coli K12 mutant carrying altered ribosomal protein (S10).

An $\text{Escherichia coli}$ mutant (JE14373) carrying decreased stability of stable RNA species was found to have altered electrophoretic mobility of a 30S ribosomal protein (S10). Recombinants covering $\text{str}$ gene (76 min on $\text{E. coli}$ linkage map by Bachmann, Low and Taylor, 1976 (ref. 1)) obtained from a cross of CSH64 × JE14373, restored normal S10 protein. The size analysis of RNAs labeled for 15 min with [³H]uridine showed 50 to 60 % decrease of 16S RNA in this mutant strain, but almost no decrease of 23S RNA at 10 or 40 min after addition of rifampicin. On the other hand, no change was observed in the stability of both rRNA pieces in its parental PA3092 strain even at 40 min after addition of rifampicin.     

A mutation in the gene for trigger factor suppresses the defect in cell division in the divE42 mutant of Escherichia coli K12

A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division. One of the suppressor mutants, designated TR4, had a novel phenotype: it was able to grow at 42°?C but not at 32°?C. The Kohara genomic library was screened for complementing clones. Clone 148 was able to complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148. DNA sequencing revealed that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation codon for this enzyme. When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32°?C but not at 42°?C, an indication that the intergenic suppressor mutation was recessive to the wild-type allele. A model is proposed that accounts for the phenotypes of the divE42 mutant and the TR4 suppressor mutant.     

Mechanism of vesicular stomatitis virus mRNA decay

The chemical and functional stability of the five vesicular stomatitis virus (VSV) messenger RNAs during infection of Chinese hamster ovary (CHO) cells was studied using the temperature-sensitive mutant, tsG114. By incubating infected cells at the nonpermissive temperature (39 °C), RNA synthesis was blocked and the five VSV mRNAs decayed chemically and functionally with a half-life of 1 to 1.5 h. However, all five VSV mRNAs were stable in vivo at 39 °C when protein synthesis was blocked with either cycloheximide or emetine. In contrast, when pactamycin was used to inhibit protein synthesis, the chemical and functional decay rates of the VSV mRNAs were indistinguishable from those observed in the absence of antibiotic. On the basis of the mode of action of each of the antibiotic inhibitors, these data imply that (a) ribosome movement along VSV mRNAs plays no role in their stabilities, and (b) each VSV mRNA contains a nuclease-sensitive site, at its 5′ end at or near the initiation site, which regulates its decay in vivo.     

Ribosomal assembly deficiency in an Escherichia coli thermosensitive mutant having an altered L24 ribosomal protein.

Localized P1 mutagenesis was used to screen for conditionally lethal mutations in ribosomal protein genes. One such mutation, 2859mis, has been mapped inside the ribosomal protein gene cluster at 72 minutes on the Escherichia coli chromosome and cotransduces at 98% with rpsE (S5). The 2869mis mutation leads to thermosensitivity and impaired assembly in vivo of 50 S ribosomal particles at 42 °C. The strain carrying the mutation has an altered L24 ribosomal protein which at 42 °C shows weaker affinity for 23 S RNA than the wild-type protein. The mutational alteration involves a replacement of glycine by aspartic acid in protein L24 from the mutant. We conclude therefore that the 2859mis mutation affects the structural gene for protein L24 (rplX).     

Studies on temperature-dependent ultraviolet light-sensitive mutants of bacteriophage T4: the structural gene for T4 endonuclease V.

Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation specifically at high temperature were isolated after mutagenesis with hydroxylamine. At 42 °C the mutants are twice as sensitive to ultraviolet light as T4D, whereas at 30 °C they exhibit survival curves almost identical to that of the wild-type strain. Complementation tests revealed that the mutants possess temperature-sensitive mutations in the v gene.Evidence is presented to show that T4 endonuclease V produced by the mutants is more thermolabile than the enzyme of the wild-type. (1) Extracts of cells infected with the mutants were capable of excising pyrimidine dimers from ultraviolet irradiated T4 DNA at 30 °C, but no selective release of dimers was induced at 42 °C. (2) Endonuclease V produced by the mutant was inactivated more rapidly than was the enzyme from T4D-infected cells when the purified enzymes were incubated in a buffer at 42 °C. From these results it is evident that the v gene is the structural gene for T4 endonuclease V, which plays an essential role in the excision-repair of ultraviolet light-damaged DNA.The time of action of the repair endonuclease was determined by using the mutant. Survival of a temperature-sensitive v mutant, exposed to ultraviolet light, increased when infected cells were incubated at 30 °C for at least ten minutes and then transferred to 42 °C. It appears that repair of DNA proceeds during an early stage of phage development.     

Cloning of the fic-1 gene involved in cell filamentation induced by cyclic AMP and construction of a delta fic Escherichia coli strain.

PA3092 is an Escherichia coli mutant that forms filaments at 43 degrees C in the presence of cyclic AMP (cAMP). The mutation responsible for this phenotype is called fic-1. We cloned fic-1 from PA3092 by selection for the neighboring argD gene. The fic-1 gene product had a relative molecular mass of 21 kilodaltons by the maxicell method. A strain with the fic gene completely deleted was constructed by replacing fic with a kanamycin resistance gene. In one of the fic-deleted strains derived from PA3092, cAMP did not induce cell filamentation at 43 degrees C, but it did in the same strain harboring a plasmid containing the fic-1 gene. These results indicate that the fic-1 gene product is necessary for the induction of cell filamentation by cAMP but is dispensable to the cell. We also found that high levels of NaCl suppressed the cell filamentation induced by cAMP.     

A mutation in Escherichia coli that mimics diauxie lag.

We have described a mutant of $\text{E.}\text{coli}$ (2S142) which shows a specific inhibition of stable RNA synthesis at 42°. The temperature sensitive lesion differs from the stringent response to amino acid starvation in that the shut off of rRNA synthesis is not associated with an inhibition of protein synthesis. The decay of ppGpp is slow at 42° with little or no pppGpp detectable. This slow decay rate is not observed in the parental strain, D10, or in 2S142 at 30°. Neither 2S142 or D10 are spoT, nor does the temperature sensitive lesion map near the spoT locus. Thus, the effect of the temperature sensitive lesion on ppGpp metabolism and rRNA synthesis seems to resemble a carbon source downshift (diauxie lag) rather than a stringent response to amino acid starvation.     

A mutation in the gene for trigger factor suppresses the defect in cell division in the divE42 mutant of Escherichia coli K12

A total of sixteen spontaneously generated, independent suppressor mutants was isolated from a mutant (divE42) of Escherichia coli K12 that is defective in cell division. One of the suppressor mutants, designated TR4, had a novel phenotype: it was able to grow at 42° C but not at 32° C. The Kohara genomic library was screened for complementing clones. Clone 148 was able to complement the mutation responsible for the cold-sensitive phenotype, and the gene for trigger factor (tig), which encodes a ribosome-associated peptidyl-prolyl cis/trans isomerase, was identified as the mutated gene by deletion analysis with the insert DNA from clone 148. DNA sequencing revealed that the mutation in the tig gene of the TR4 suppressor mutant was a single nucleotide insertion (+A) at a distance of 834 nucleotides from the initiation codon for this enzyme. When the wild-type tig gene was introduced into the TR4 suppressor mutant, the bacteria were able to grow at 32° C but not at 42° C, an indication that the intergenic suppressor mutation was recessive to the wild-type allele. A model is proposed that accounts for the phenotypes of the divE42 mutant and the TR4 suppressor mutant. Received: 3 March 1998 / Accepted: 14 July 1998     

UBQLN2 undergoes a reversible temperature-induced conformational switch that regulates binding with HSPA1B: ALS/FTD mutations cripple the switch but do not destroy HSPA1B binding

Here we present evidence, based on alterations of its intrinsic tryptophan fluorescence, that UBQLN2 protein undergoes a conformational switch when the temperature is raised from 37 °C to 42 °C. The switch is reset on restoration of the temperature. We speculate that the switch regulates UBQLN2 function in the heat shock response because elevation of the temperature from 37 °C to 42 °C dramatically increased in vitro binding between UBQLN2 and HSPA1B. Furthermore, restoration of the temperature to 37 °C decreased HSPA1B binding. By comparison to wild type (WT) UBQLN2, we found that all five ALS/FTD mutant UBQLN2 proteins we examined had attenuated alterations in tryptophan fluorescence when shifted to 42 °C, suggesting that the conformational switch is crippled in the mutants. Paradoxically, all five mutants bound similar amounts of HSPA1B compared to WT UBQLN2 protein at 42 °C, suggesting that either the conformational switch is not instrumental for HSPA1B binding, or that, although damaged, it is still functional. Comparison of the poly-ubiquitin chain binding revealed that WT UBQLN2 binds more avidly with K63 than with K48 chains. The avidity may explain the involvement of UBQLN2 in autophagy and cell signaling. Consistent with its function in autophagy, we found UBQLN2 binds directly with LC3, the autophagosomal-specific membrane-tethered protein. Finally, we provide evidence that WT UBQLN2 can homodimerize, and heterodimerize with WT UBQLN1. We show that ALS mutant P497S-UBQLN2 protein can oligomerize with either WT UBQLN1 or 2, providing a possible mechanism for how mutant UBQLN2 proteins could bind and inactivate UBQLN proteins, causing loss of function.