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.     

YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes

In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl–tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.     

Bacteriophage T4 mutants deficient in alteration and modification of the Escherichia coli RNA polymerase

An extensive screening of coliphage T4 mutants has revealed two distinct classes defective, respectively, in the two sequential phage-induced phosphorylations of the host RNA polymerase, alteration and modification. The existence of these mutants proves that T4-specified functions are involved in both processes. The viabilities of these mutants demonstrate that neither alteration nor modification is essential for growth in Escherichia coli B/r. Physiological studies after infection of E. coli B/r have failed to reveal any abnormalities of phage deficient in alteration or modification. Both mutants normally inhibit host protein and stable RNA synthesis and normally express all classes of T4 genes. Thus, these specific phage-induced structural changes in the host RNA polymerase are not fundamental to the control of gene expression during T4 development. Alteration and modification may be required for growth in some strains of E. coli and hence be selectively advantageous because they extend the normal host range of the phage.Alteration appears to be catalyzed by a T4 function injected with the DNA. A polypeptide of molecular weight 61,000, which is probably cleaved during morphogenesis from a precursor of molecular weight 79,000, is missing in phage particles of alteration-deficient strains and may be the phage activity so injected. The T4 gene involved in alteration is named alt.Modification is controlled by a T4-replicative gene that has been mapped into a region of about 500 base-pairs between genes 39 and 56. These mapping data show that the defect in α modification defines a new T4 gene, named mod.     

A novel Escherichia coli mutant capable of DNA replication in the absence of protein synthesis.

An Escherichia coli mutant capable of continued DNA synthesis in the presence of chloramphenicol has been isolated by an autoradiographic technique. The DNA synthesis represents semiconservative replication of E. coli DNA. It can occur in the presence of chloramphenicol or in the absence of essential amino acids, but not in the presence of an RNA synthesis inhibitor, rifampin. The mutant, termed constitutive stable DNA replication (Sdr^c) mutant, appears to grow normally at 37 °C with a slightly slower growth rate than that of the parental strain. DNA replication in the mutant occurs at a reduced rate after 60 minutes in the absence of protein synthesis and continues linearly for several hours thereafter. This distinct slowdown in the DNA replication rate is due to a reduced rate of DNA synthesis in all the cells in the population. Constitutive stable DNA replication appears to require the dnaA and dnaC gene products. The sdr^c mutation has been mapped near the pro-lac region of the E. coli chromosome. The mutation is recessive. Autoradiographic experiments have ruled out the possibility of multiple initiations during a cell cycle. The implication of the above findings is discussed in terms of the regulation of chromosome replication in E. coli.     

Protein synthesis in Bacillus subtilis. II. Selective translation of natural mRNAs and its possible relation to the species-specific inhibition of protein synthesis by lincomycin and erythromycin.

Vacant ribosomal couples from Bacillus subtilis W168 incorporate only very small amounts of amino acids into polypeptides in response to Escherichia coli cellular RNA or bacteriophage f2 RNA, but are observed to form initiation complexes in the presence of f2 RNA. Vacant ribosomal couples from E. coli acquire pressure-resistance, but do not bind fMet-tRNA, when incubated with B. subtilis RNA in the absence of ribosomal wash fraction. The implied mRNA binding in the absence of salt wash fraction, taken with previously reported observations of salt wash-independent translation of mRNAs from Grampositive bacteria, suggests that mRNAs from Gram-positive bacteria have an active functional character which is masked or absent in mRNAs from Gram-negative sources. It is proposed that this property of B. subtilis mRNAs is required by B. subtilis ribosomes for some translational function subsequent to the formation of the 70 S initiation complex, and that f2 RNA, while it is bound by B. subtilis ribosomes in initiation complexes, is not translated because it lacks this feature.The antibiotic lincomycin has been found to inhibit translation of natural mRNAs in vitro in systems from Gram-positive bacteria at concentrations 10 to 100 times lower than those necessary to inhibit translation in systems from Gram-negative species. Lincomycin does not inhibit formation of initiation complexes by vacant couples from B. subtilis or E. coli. Taken with the published findings of other investigators, these results are interpreted as indicating that the first translocation step following assembly of the initiation complex may coincide with a transition between distinct “initiating” and “elongating” states of the ribosome, and that this transition may involve structural elements, and possibly mechanisms, which are different in Gram-positive systems than in Gram-negative systems.A comprehensive model is constructed to account for the results of these studies and for the published findings of other investigators. It is proposed that some feature of Gram-positive mRNA, perhaps a vestige of early protein synthetic systems, is required by the ribosomes of Gram-positive bacteria to facilitate the transition between initiating and elongating ribosomal states. Inhibition of protein synthesis by lincomycin and the similarly species-specific macrolide antibiotic erythromycin is interpreted as an allosteric effect on the transition between initiating and elongating ribosomal states, in which the different binding affinities of ribosomes from Gram-positive and Gram-negative bacteria for the drugs are related to the functional differences between the two types of systems at this critical step. The implications of this interpretation of interspecies translational specificity for mechanisms of translational control in the cell and for the nature of the divergence of bacterial protein synthesis systems into Gram-positive and Gram-negative types are discussed.     

Protein synthesis in Bacillus subtilis. I. Hydrodynamics and in vitro functional properties of ribosomes from B. subtilis W168.

On the basis of their sedimentation properties, the ribosomal particles in crude extracts of Bacillus subtilis W168 are characterized as pressure-sensitive couples, pressure-resistant couples, or non-associating subunits. Pressure-sensitive couples dissociate into subunits, yielding a peak at 60 S in the gradient profile, on sedimentation at high speed in the presence of 10 to 15 mm-Mg²⁺. Under the same conditions, pressure-resistant couples sediment at 70 S. Under certain conditions, pressure-resistant couples apparently aggregate, possibly in 70 S · 70 S dimers. Procedures are described for the isolation of pressure-sensitive couples from B. subtilis. The isolated couples are shown by chemical fixation experiments to require approximately twice the Mg²⁺ concentration required by Escherichia coli couples to remain associated at atmospheric pressure.All three types of B. subtilis ribosome incorporate amino acids into acid-insoluble material in the presence of B. subtilis cellular RNA, B. subtilis ribosomal salt wash fraction, and E. coli post-ribosomal supernatant. Overall incorporation, dependence on added RNA, and dependence on salt wash fraction are greatest with pressure-sensitive couples. The products of protein synthesis in vitro stimulated by total B. subtilis RNA appear to be a low molecular weight subset of the proteins synthesized most abundantly in vivo. Incubation of pressure-sensitive couples with cellular RNA from B. subtilis, fMet-tRNA_f^Met, ribosomal salt wash fraction and GTP results in their conversion to pressure-resistant couples, with concomitant and stoichiometric binding of fMet-tRNA to the 70 S species. It is concluded that in B. subtilis as in E. coli, pressure-sensitive couples are “vacant”, while pressure-resistant couples are “complexed” with messenger RNA. fMet-tRNA-bearing complexed couples are interpreted as initiation complexes in which ribosomes have bound mRNA, presumably at initiation sites. Their formation in vitro is strictly dependent on RNA, salt wash fraction and fMet-tRNA when vacant ribosomal couples are used.     

T4 phage and T4 ghosts inhibit f2 phage replication by different mechanisms

Both T4 phage and DNA-free ghosts inhibit replication of RNA phage f2. Most but not all of the effects by T4 upon f2 growth can be blocked by the addition of rifampicin prior to T4 superinfection; by contrast, the inhibition of f2 synthesis by T4 ghosts cannot be blocked by rifampicin. This indicates that inhibition by intact T4 requires gene function, while inhibition by ghosts does not. There is a small, multiplicity-dependent inhibition by viable T4 on f2 growth in the presence of rifampicin which may be similar to the gene function-independent inhibition by T4 ghosts. With one viable T4 per cell, there appears to be no effect by viable T4 upon f2 growth which does not require T4 gene action. Moreover, increasing multiplicities of viable T4 appear to inhibit T4 replication as well.In the absence of rifampicin, pre-existing f2 single and double-stranded RNA are degraded after superinfection by viable T4, but remain stable after superinfection by ghosts. However, no new f2 RNA is synthesized after superinfection with either. In the presence of rifampicin, f2-specific protein synthesis is largely unaffected by viable T4, but is completely inhibited by ghosts. Both Escherichia coli, as well as f2-speciflc polysomes disappear in the presence of ghosts.We conclude that, at low multiplicities, T4 phage and T4 ghosts inhibit replication of f2 phage, and presumably host syntheses, by different mechanisms.     

Isolation of Escherichia coli mRNA and Comparison of Expression Using mRNA and Total RNA on DNA Microarrays

Bacterial messenger RNA (mRNA) is not coherently polyadenylated, whereas mRNA of Eukarya can be separated from stable RNAs by virtue of polyadenylated 3′-termini. We have developed a method to isolate Escherichia coli mRNA by polyadenylating it in crude cell extracts with E. coli poly(A) polymerase I and purifying it by oligo(dT) chromatography. Differences in lacZRNA levels were similar with purified mRNA and total RNA in dot blot hydridizations for cultures grown with or without gratuitous induction of the lactose operon. More broadly, changes in gene expression upon induction were similar when cDNAs primed from mRNA or total RNA with random hexanucleotides were hydridized to DNA microarrays for the E. coli genome. Comparable signal intensities were obtained with only 1% as much oligo(dT)-purified mRNA as total RNA, and hence in vitro poly(A) tailing appears to be selective for mRNA. These and additional studies of genome-wide expression with DNA microarrays provide evidence that in vitro poly(A) tailing works universally for E. coli mRNAs.     

Characterization of Late Polyoma mRNA

Polyoma-infected mouse kidney cell cultures were labeled with [³H]uridine for 3 h late in the lytic cycle (26 to 29 h after infection) and RNA was extracted from cytoplasm and nuclei and from isolated polyribosomes. Sedimentation velocity analysis in sucrose gradients showed that polyoma-specific “giant” and 26S RNAs are present exclusively in the nucleus. RNA associated with cytoplasmic polyribosomes was analyzed by sedimentation in aqueous sucrose density gradients and dimethylsulfoxide sucrose gradients, as well as by polyacrylamide gel electrophoresis. Polyoma-specific RNA in polyribosomes consists of at least two classes, with sedimentation coefficients of 16 (major fraction) and 19S (minor fraction) in aqueous sucrose gradients and 15 and 17S, respectively, in dimethylsulfoxide gradients. Estimates based on dimethylsulfoxide gradient and analysis suggest a molecular weight of approximately 500,000 for 16S RNA and 700,000 for 19S RNA. These polyoma RNAs seem to undergo reversible conformational changes under the different conditions of analysis. We cannot exclude the possibility that they contain more than one molecular species.     

Cistron Specific Translation Control Protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

GENE expression may be controlled during translation by ribosomal selection of mRNAs or even individual cistrons. Escherichia coli initiation factors associated with ribosomes affect the binding of ribosomes to mRNA^1,2; initiation factor IF3, for instance, influences the specificity of mRNA-ribosome interaction^3,4. IF3 activity has been separated into several fractions which show various specificities for different mRNA cistrons^4–9. An important problem is the possibility of intracellular changes in IF3 activity^10–12. From uninfected E. coli, we have now isolated a protein which changes the specificity of IF3 toward different mRNAs; we call this interference factor i. Pure factor i binds to IF3 and specifically affects the translation of T4 and MS2 RNA. Whereas the initiation of translation of the MS2 coat protein cistron is inhibited by factor i, the synthetase cistron—when available—is more rapidly initiated in the presence of factor i. The overall translation of T4 mRNA appears unchanged by factor i, but certain cistrons are stimulated at the expense of others. Interfering factors such as factor i could be important in controlling translation in E. coli.     

Inactivation of SSM4, a new Saccharomyces cerevisiae gene,suppresses mRNA instability due to rna14 mutations

Decay rates of mRNAs depend on many elements and among these, the role of the poly(A) tail is now well established. In the yeast Saccharomyces cerevisiae, thermosensitive mutations in two genes, RNA14 and RNA15, result in mRNAs having shorter poly(A) tails and reduced half-life. To identify other components interacting in the same process, we have used a genetic approach to isolate mutations that suppress the thermosensitivity of an rna14 mutant strain. Mutations in a single locus, named SSM4, not only suppress the cell growth phenotype but also the mRNA instability and extend the short mRNA poly(A) tails. The frequency of appearance and the recessive nature of these mutations suggested that the suppressor effect was probably due to a loss of function. We failed to clone the SSM4 gene directly by complementation, owing to its absence from gene banks; it later emerged that the gene is toxic to Escherichia coli, but we have nevertheless been able to clone the SSM4 sequence by Ty element transposition tagging. Disruption of the SSM4 gene does not affect cell viability and suppresses the rna14 mutant phenotypes. The protein encoded by the SSM4 gene has a calculated molecular mass of 151 kDa and does not contain any known motif or show homology with known proteins. The toxicity of the SSM4 gene in E. coli suggests that a direct biochemical activity is associated with the corresponding protein.     

Purification and properties of two messenger-discriminating species of E. coli initiation factor 3

Two messenger-discriminating species of the E. coli initiation factor 3, previously isolated by us, have been purified to near homogeneity as judged by disc electrophoresis and electrofocusing on polyacrylamide gels. One of the two species, IF3α, is highly specific for MS2 phage, E. coli, and early T4 RNA and has low activity with late T4 phage RNA, whereas the reverse is true of the other species, IF3β. Both have the same level of ribosome dissociation factor (DF) activity. IF3α and IF3β seem to consist of a single polypeptide chain weighing 23,000–23,500 and 21,000–21,500 daltons, respectively. Electrophoretic analysis of the phage-specific proteins synthesized by the cell-free translation of MS2 RNA, with either IF3α or IF3β, shows that these molecular species of IF3 either have no cistron discrimination ability or do not differ in this regard.     

Gene cloning and soluble expression of Aspergillus niger phytase in E. coli cytosol via chaperone co-expression

A phytase gene from Aspergillus niger was isolated and two Escherichia coli expression systems, based on T7 RNA polymerase promoter and tac promoter, were used for its recombinant expression. Co-expression of molecular chaperone, GroES/EL, aided functional cytosolic expression of the phytase in E. coli BL21 (DE3). Untagged and maltose-binding protein-tagged recombinant phytase showed an activity band of ~49 and 92 kDa, respectively, on a zymogram. Heterologously-expressed phytase was fractionated from endogenous E. coli phytase by (NH₄)₂SO₄ precipitation. The enzyme had optimum activity at 50 °C and pH 6.5.     

Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sp. strains

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bongori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperature phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.     

Absence of a 5S RNA Component in the Mitochondrial Ribosomes of <Emphasis Type="Italic">Neurospora crassa</Emphasis>

RIBOSOME-BOUND, low molecular weight RNA, distinct from tRNA, was first observed in E. coli by Rosset and Monier¹. This RNA, which has a sedimentation coefficient of about 5S, is now considered to be a universal component of ribosomes. We report here our attempts to find low molecular weight RNAs other than tRNA in mitochondria of Neurospora. Our evidence suggests that the mitochondrial ribosomes of this organism lack a 5S RNA component.