Cyrus Levinthal

Protein synthesis by Escherichia coli infected with bacteriophage T4D

Abstract Proteins made by E. coli cells infected with bacteriophage T4 were analyzed by a method which combined disc electrophoresis and autoradiography. The precursorproduct relationship between subunits and larger components (head, tail) was studied by taking advantage of the fact that the larger components cannot penetrate into the gel used in disc electrophoresis. These studies have shown that the early proteins are not controlled as a single homogeneous class all members of which are synthesized during the same time periods; instead, they start being formed and are shut off at various times during the early part of the infection process. Amber mutants in gene 30 (polynucleotide ligase-defective) synthesized a small amount of DNA, which was later degraded to a fraction soluble in trichloroacetic acid. The ligase-defective mutants were capable of synthesizing an almost normal amount of late proteins, whereas all other DNA-negative mutants, including a deoxycytidine triphosphatase(dCTPase)-defective mutant and maturation-defective mutants could not induce late protein synthesis. The dCTPase-defective mutant, which also synthesizes a small amount of unstable DNA, did not induce late protein synthesis even when the degradation of DNA was prevented.

Hybrid protein formation of E. coli alkaline phosphatase leading to in vitro complementation.

In vitro complementation has been demonstrated for alkaline phosphatase-negative mutants of E. coli . Enzymically active alkaline phosphatase can be formed by mixing monomer subunits derived from inactive purified proteins which are antigenically related to the wild-type enzyme of E. coli . Preparations of these proteins purified from four different phosphatase-negative mutants of E. coli could be reacted in pairs to yield partially active enzyme. Experimental evidence indicates that the active protein is a hybrid molecule composed of a monomer from each of the mutant proteins used in the reaction. The monomers, which canJse prepared from the native protein by mild acid treatment or reduction with thioglycollate in urea, undergo a temperature and metal-dependent bimolecular reaction to yield a product distinct from normal enzyme but with partial enzymic activity. The normal alkaline phosphatase protein is composed of two identical subunits whose structure is determined by a single functioning genetic unit. These results thus support the theory of hybrid protein formation which has been proposed to account for intra-cistron complementation.

Messenger RNA decay and protection

The decay of messenger RNA in Bacillus subtilis has been studied under a variety of conditions which interfere with protein synthesis. Changes in the growth rate produced by altering the medium do not affect the proportion of messenger relative to total RNA. The decay rate has a large temperature coefficient varying roughly as the rate of protein synthesis. However, the decay does not depend on protein synthesis since it occurs normally in the presence of puromycin. Energy starvation produced by anaerobiosis, cyanide or azide markedly protects the messenger from its normal decay as does chloramphenicol. Puromycin will release this protection and restore rapid decay. It is suggested that the messenger is protected by virtue of being held on to a ribosome and the release of the protection occurs when the messenger is released from the ribosome.

Measurement of the unstable RNA in exponentially growing cultures of Bacillus subtilis and Escherichia coli

Abstract A new technique for measuring the rate of turnover and quantity of unstable RNA in exponentially growing cells has been developed which escapes the interpretive difficulties of earlier methods. It depends upon the fact that an unstable RNA fraction has easily measurable effects upon the kinetics of labelling of GTP; before the GTP pool can become fully labelled, unlabelled nucleotides present as GTP or in unstable RNA must be washed into stable RNA. (There is no equilibration with guanine in the medium.) Consequently, if the uptake of label into GTP is rapid, with only the delay predicted from the experimentally measured GTP pool size, then it is possible to conclude that there is little or no unstable RNA. In Bacillus subtilis, however, the GTP pool is labelled very slowly which, with other data, indicates a large amount of unstable RNA (9% of the guanine residues of the cell in unstable RNA). Similar measurements of the rapidity of GTP labelling in Escherichia coli indicate that there is much less unstable RNA (about 3%) in this organism. The curve expressing GTP specific activity as a function of time is shown to be the sum of two exponential terms with very different time constants. From the relative sizes of the two exponential terms, it is possible to estimate the rate of turnover of the unstable RNA. The result is weighted most heavily for the slow components of the decaying messenger, and thus it sets an upper limit to the true average decay time for the unstable RNA. This decay time (upper limit) was three minutes for the unstable RNA in B. subtilis grown at 37 °C with a doubling time of 115 minutes and six minutes when the cells were grown at 30 °C with a doubling time of 225 minutes. For E. coli (growing at 30 °C with a doubling time of 66 minutes) the decay time measured in this way was about four minutes. Thus the decay times as measured in these experiments are the same order of magnitude as have been observed by measuring the decay of pulse-labelled RNA and the decay of protein synthetic capacity after the addition of actinomycin. The various estimates of the decay rate of messenger RNA in bacteria are discussed in terms of the possible theoretical ambiguities in the interpretation of each.

RNA metabolism in T4-infected Escherichia coli

Abstract Discrete species of 14 C-labeled messenger RNA from T4-infected Escherichia coli can be detected as bands on polyacrylamide gels after electrophoresis and autoradiography. Experiments are presented which demonstrate that the transcriptional patterns change during the course of infection and that protein synthesis inhibition at appropriate times of infection prevents certain transcriptional transitions. It is shown that the synthesis of stable host RNA (23 s, 16 s, 5 s and transfer RNA) ceases after two minutes of infection at 25 °C but that the addition of chloramphenicol to the culture before infection prevents this shut off. “Early” phage RNA which is synthesized before the onset of DNA synthesis can be divided into two subclasses by virtue of the fact that only one class is synthesized in chloramphenicol pretreated cultures. Also, chloramphenicol prevents the synthesis of late phage RNA even if added at a time when it allows a significant amount of phage DNA to be synthesized. This indicates that phage DNA synthesis is a necessary but not a sufficient requirement for transcription of the late phage genes. Analysis of the RNA synthesized by cultures infected with various phage mutants demonstrates that phage DNA synthesis and the functioning of genes 55 and 33 are required for the synthesis of late messenger RNA which is synthesized after 20 minutes of infection at 25 °C. The striking similarity of the RNA patterns from cultures infected with amber mutants in genes 33 and 55 corroborates their classification in the same class of maturation defectiveness. It is shown that though almost all of the RNA labeled during phage infection is unstable, several RNA species in the molecular weight range 20,000 to 50,000 are stable and accumulate in large amounts during infection. The results are discussed in relation to our current understanding of the mechanisms controlling RNA and protein synthesis in phage-infected cells.

Messenger RNA and RNA transcription time.

The properties of unstable RNA in Bacillus subtilis have been studied under a variety of conditions. It was found that: (1) The unstable fraction constitutes approximately 8% of the total RNA in Bacillus subtilis . The corresponding proportions in two strains of Bacillus megaterium are 6 and 9% of the total RNA. (2) After short pulses of radioactive uracil, most of the labeled RNA is distinguished from 16 and 23 s ribosomal, and from soluble RNA, by its sedimentation properties. (3) Completed 16 and 23 s ribosomal RNA molecules are stable in the presence of actinomycin. If there are ribosomal RNA molecules which are unfinished at the time actinomycin is added, they are degraded. (4) After a 20 to 30-second pulse of radioactive uracil, about 50% of the incorporated label is found to sediment with 70 s ribosomes and polysomes. When actinomycin is added to the medium, the degradation of the unstable RNA is accompanied by the disappearance of polyribosomal structures. (5) If the polysomes are separated by shearing forces, the unstable RNA originally associated with polysomes sediments with 70 s ribosomes. Using this technique, it has been estimated that in polyribosomes there are, on the average, 150,000 daltons or 500 nucleotides of messenger RNA per 70 s ribosomes. (6) After a 30-second pulse of radioactive uracil and subsequent incubation in actinomycin. the radioactivity stable to degradation is found entirely in 30 and 50 s ribosomal subunits and in soluble RNA. The specific activity of the 50 s particle is less than that of the 30 s particle and of soluble RNA. If the length of the pulse of radioactive precursors is increased to three minutes, then all the stable fractions have approximately the same specific activity after incubation in actinomycin. Possible explanations of this result are discussed. It is suggested that the transcription of RNA on the DNA template may be a slow process requiring 1 min to synthesize 1·5 to 2·0 x 10 6 daltons of RNA.

Effects of λ-phage infection on bacterial synthesis

Abstract RNA and protein synthesis in λ infected cells have been studied. A marked depression of the synthesis of these macromolecules was observed, and was found to depend on the multiplicity of infection. This effect occurs in sensitive cells as well as in lysogenic immune cells. Depression of RNA synthesis takes place even if the cells have been pretreated with chloramphenicol and this depression seems to affect all molecular species of RNA equally. The types of newly synthesized proteins are substantially similar to those of the uninfected control. Host restricted λ or ghosts of phage λ do not show this effect, suggesting that the effects are not due to a protein component of the phage itself.

Isolation and characterization of complementation products of Escherichia coli alkaline phosphatase

Hybrid dimers of the Escherichia coli alkaline phosphatase were formed in vitro with one subunit each from various mutants. When one of the subunit species was labeled for density (D, 15N) and for radioactivity, it was possible to isolate the hybrids and study their properties directly. Such studies showed that some subunit species combine with greater affinity than others and that different hybrids can have different specific enzymic activities. The genetic map for the mutants studied is presented and its relationship to complementation is discussed.