T4 mRNA Research Articles

Autoregulation of gene expression: Quantitative evaluation of the expression and function of the bacteriophage T4 gene 32 (single-stranded DNA binding) protein system

The free concentration of bacteriophage T4-coded gene 32 (single-stranded DNA binding) protein in the cell is autoregulated at the translational level during T4 infection of Escherichia coli. The control of the synthesis of this protein reflects the following progression of net (co-operative) binding affinities for the various potential nucleic acid binding targets present: single-stranded DNA > gene 32 mRNA > other T4 mRNAs ⩾ double-stranded DNA. In this paper we show that the free concentration of gene 32 protein is maintained at 2 to 3 μ m, and use the measured binding parameters for gene 32 protein, extrapolated to intracellular conditions, to provide a quantitative molecular interpretation of this system of control of gene expression. These results are then further utilized to define the specific autoregulatory binding sequence (translational operator site) on the gene 32 mRNA as a uniquely unstructured finite binding lattice terminated by elements of secondary structure not subject to melting by gene 32 protein at the autoregulated concentration, and to predict how this site must differ from those found on other T4 messenger RNAs. It is shown that these predictions are fully consistent with available T4 DNA sequence data. The control of free protein concentration as a method of genome regulation is discussed in terms of other systems to which these approaches may apply.

Initiation factor-independent translation of mRNAs from Gram-positive bacteria.

Initiation factor-independent translation of mRNA derived from bacillus phage phi29 DNA occurs with translation systems derived from Bacillus subtilis or Escherichia coli. This is in sharp contrast to the strict dependence on ribosome salt wash fraction of E. coli ribosomes for the translation of T7 and other mRNAs derived from Gram-negative organisms.

Translational regulation: identification of the site on bacteriophage T4 rIIB mRNA recognized by the regA gene function.

The bacteriophage T4 gene regA encodes a protein that diminishes the expression of many unlinked early T4 genes. Previous work demonstrated that regA-mediated repression occurs after transcription. We report here on the identification of the target site on one regA-sensitive mRNA, the message encoding the phage T4 rIIB protein. The target for regA-mediated action overlaps the translational initiation domain of the rIIB messenger. The regA protein may be a repressor that operates translationally on a significant and interesting set of early phage T4 mRNAs.

Regulation of the synthesis of the T4 DNA polymerase (gene 43)

The product of T4 gene 43, DNA polymerase (P43), regulates the level of its own synthesis. Normally, the rate of P43 synthesis declines rapidly 10 min after infection. However, P43 synthesis can be induced late in infection by shifting cells infected with temperature sensitive mutants of P43 from 30 to 42° ( Russel, 1973). Studies reported here show that induction of P43 synthesis late in infection also occurs after shifting several different amber or temperature sensitive T4 mutant-infected cells from 30 to 42°; the mutations causing the effect are in T4 genes essential for DNA replication. This turn on can be 5- to 10-fold greater than the level of P43 synthesis during comparable wild-type infections. In addition to P43, several other proteins, including P41, are turned on in these experiments. Other observations relevant to this phenomenon are as follows. First, blocking DNA synthesis with chemical inhibitors does not enhance the rate of P43 synthesis. Second, the increased expression of P43 after the temperature shift is dependent on new RNA synthesis; i.e., the increased expression is blocked by the addition of rifampicin and does not occur when the host cell RNA polymerase is temperature sensitive or when the infecting phage contains a tsG1 mutation in the T4 mot locus, thought to be necessary for normal P43 synthesis. Furthermore, hybridization studies of labeled T4 mRNA with plasmid DNA (pVH18), containing the middle one-third of gene 43, show that new P43 mRNA is made when the increased expression of P43 occurs. Third, unlike the expression of P32, the increased expression of P43 is not affected by the absence of P46-mediated gaps in replicative DNA. Fourth, studies examining the stability of P43 under a variety of conditions indicate that P43 is not degraded at 42°. Several models of the control of gene 43 expression are discussed.

Radiation sensitivity of messenger RNA

Messenger RNA function is inactivated by irradiation with ultraviolet light. A unit length mRNA (in bases) is 2-3 times more sensitive than a unit length of DNA (in base pairs) with respect to the inactivation of template function. These data stem from four experimental systems all of which do not repair DNA: the translation of E. coli mRNA in rifampicin-treated cells, of T7 mRNA in infected E. coli, of f2 phage RNA in vivo, and of stable mRNA in chromosomeless minicells. The comparison of relative sensitivities to UV is relevant to the technique of UV mapping of transcription units which enjoys increasing popularity in pro- and eukaryotic genetic research.

Regulation of ribosome function following bacteriophage T4 infection

The rate of translation in bacteriophage T4-infected Escherichia coli has been studied. It was observed that at about ten minutes after infection at 37 °C the rate of protein synthesis declines to 40 to 50% of the rate observed during the first ten minutes, yet all cells remain intact for at least 60 minutes. This drop in the rate of general protein synthesis is correlated with a change in the ability of initiation factor-free ribosomes to translate both global T4 messenger RNAs and a specific T4 messenger, deoxynucleotide kinase (EC 2.7.4.4) mRNA. The alteration in ribosome function begins between five and ten minutes after infection and minimum ribosome activity is reached at approximately 20 minutes after infection. A late T4 gene is involved, as shown by the fact that the alteration in ribosome function is not observed in amB1292-infected cells (i.e. cells which synthesize early but not late T4 mRNAs).

An exoribonuclease from [formula omitted]: Effect of modifications of 5′ end groups on the hydrolysis of substrates to 5′ mononucleotides

Using poly(A) as a substrate, an exoribonuclease has been purified from the high-salt wash of ribosomes of Saccharomyces cerevisiae . The product of the reaction of the exoribonuclease is 5′ AMP. Hydrolysis of [ 3H](pA) 3[ 14C](pA) n shows that both labels are released at the same rate, suggesting that the enzyme acts in a processive manner. Removal of the terminal phosphate of poly(A) with alkaline phosphatase reduces the rate of hydrolysis by 80%. Treatment of the terminally dephosphorylated poly(A) with polynucleotide kinase restores the activity. Two 5′ capped mRNA's have been tested and they are hydrolyzed slowly, if at all, by the enzyme. In contrast, phage T4 mRNA, ribosomal RNA, and encephalomyocarditis viral RNA are hydrolyzed at greater than 50% of the rate of poly(A).

Regulation of transcription of the late genes of bacteriophage T7.

The transcription program of bacteriophage T7 in vivo was analyzed by hybridizing T7 mRNAs, labeled at intervals after infection, to Hpa I restriction fragments of T7 DNA. Transcripcion of the late genes is temporally regulated: class II genes are transcribed between 4 and 16 min after infection; most class III genes are transcribed from 8 min after infection until lysis. Genes 8--10 are transcribed as both class II and class III genes. The rate of T7 RNA synthesis decreases sharply at 10 min after infection. The rapid decrease in the rate of T7 RNA synthesis and the shutoff of class II RNA synthesis were not observed in cells infected with phage defective in gene 3.5 (lysozyme). Although the decrease in the rate of T7 RNA synthesis is independent of DNA replication, the failure to shut off class II RNA synthesis normally in 3.5-- -infected cells may reflect the role of T7 lysozyme in DNA replication. In vitro, the regions of T7 DNA transcribed by the phage RNA polymerase were found to be dependent upon ionic conditions.

Purification and some novel properties of Escherichia coli RNase II.

RNase II of Escherichia coli (EC 3.1.4.23) has been purified to apparent homogeneity. The K+-activated diesterase activity against poly(U), which defines RNase II, cochromatographs with activity against T4 mRNA or pulse-labeled E. coli RNA successively on DEAE-cellulose, hydroxyapatite or phosphocellulose, and Sephadex G-150 columns. Activities with both substrates are selectively reduced to less than 2% of the wild type level in a newly isolated mutant strain, S296, or after thermal inactivation in a mutant strain with temperature-sensitive RNase II. RNase II releases 5'-XMP without a lag as its only detectable alcohol-soluble produce from all substrates and has an apparent molecular weight of 80,000 to 90,000 in both nondissociating and sodium dodecyl sulfate-polyacrylamide gels. The pure enzyme shows the standard K+ activation against poly(A), poly(U), or poly(C), but only a slight preference for K+ over Na+ ions with T4 mRNA or pulse labeled E. coli RNA as substrate. Uniformly labeled E. coli rRNA or tRNA is degraded little if at all.

Role of potassium in the synthesis and decay of two specific bacteriophage T4 messages

The role of K+ in the in vivo metabolism of specific phage T4 messengers was studied. By using a mutant of Escherichia coli defective in its ability to accumulate K+ from the growth medium, it was possible to rapidly deplete cells of their intracellular K+ and in this way determine K+-dependent reactions in vivo. The rate constants for accumulation, synthesis, and decay of the early enzymes deoxynucleotide kinase and alpha-glucosyl transferase were determined. It was shown that there is a very close association between mRNA synthesis and its decay, indicating that a mechanism may be present in the cell that can regulate the concentration of these RNAs. Since the mRNA's for these enzymes are very stable in cells depleted of K+, K+ depletion may be a useful method for the isolation of functional T4 mRNA.

Use of antibiotics to determine ribosome-messenger RNA interactions necessary for in vivo stability of specific messengers

With the use of inhibitors of individual reactions in protein synthesis, a method has been developed for (a) determining the role of ribosome messenger RNA interactions in specific bacteriophage T4 mRNA stability and (b) localizing the primary site of interaction of messenger ribonuclease (mRNase) on messengers. Antibiotics that freeze ribosomes in or near the initiation site stabilize T4 deoxynucleotide kinase mRNA. In contrast, T4 α-glucosyltransferase mRNA is stable only when the polysome configuration is kept intact. These results indicate the deoxynucleotide kinase mRNA initiation site is most susceptible to mRNase action, whereas the primary site of mRNase action on the α-glucosyltransferase mRNA is distal to the initiation site. Determining the role of ribosome-mRNA interactions in mRNA stability by the use of specific inhibitors of protein synthesis may be applicable to any procaryotic or eucaryotic mRNA that can be translated in vitro.

Bacteriophages T7 and T3 as Model Systems for RNA Synthesis and Processing

This chapter reviews that in Escherichia coli , site-specific cleavages by RNase III are the initial events in the processing of rRNAs and are also responsible for generating the individual “early” mRNAs of bacteriophages T7 and T3. Using purified E. coli RNA polymerase and RNase III, it is possible to synthesize in vitro early T7 and T3 mRNAs that are identical to those found in infected cells. RNase III cleavage of rRNA and early mRNA takes place at specific processing signals that are present in the RNA itself. It discusses that enzymes similar to E. coli RNase III might be found in other organisms. One approach to identifying a RNase-III-type enzyme in a source other than E. coli is to ask whether extracts prepared from the organism contain an activity that will faithfully process T7 and T3 early RNAs. A complementary approach would be to test a putative precursor RNA with RNase III from E. coli to see whether the RNA contains processing signals and whether specific products are generated. The chapter summarizes some aspects of T7 and T3 early RNA processing and the properties of E. coli RNase III that is useful in studies of this type.

Translation of bacteriophage T4 mRNA into active lysozyme by a wheat germ cell-free system

T4 bacteriophage mRNA for lysozyme was extracted from T4 phage infected E. coli cells, partially purified by column chromatography, and translated in a heterologous cell-free protein synthesizing system prepared from wheat germ. The translation product was confirmed by SDS polyacrylamide gel electrophoresis and enzymatic activity — bacteriolysis as tested with Micrococcus luteus . The specific activity of the enzyme prepared was 660 U/mg.

Photo-affinity labeling of 23 S rRNA by an analog of fMet-tRNA Metf

Summary The technique of photo-affinity labeling has been employed to localize ribosomal components at the peptidyl-transferase center that interact with the initiator tRNA. The photo-sensitive p-azido-N-tBoc -Phe-(35S)Mot-tRNAMetf acts as an analog of fMet-tRNAMetf as it requires initiation factors and T4 mRNA for binding to 70 S ribosomes. Irradiation of the reversible binding complex results in a covalent linkage of the label to the ribosome, mostly to 23 S rRNA and specifically to its 18 S fragment. This is the first instance where an analog of the initiator tRNA has been shown to interact with 23 S rRNA, a ribosomal component likely to be involved in the interaction of donor substrates with the peptidyl transferase center.

The distribution of functional bacteriophage T4 mRNA on polysomes

Functional bacteriophage T4 deoxynucleotide kinase and α-glucosyl transferase mRNAs can be isolated from polysomes extracted from cells 8 min after infection. At least 55% of the 8-min deoxynucleotide kinase mRNA is associated with polysomes and is released from the cell membrane by deoxyribonuclease (DNase) treatment (soluble mRNA). Approximately 20% of the kinase mRNA remains tightly bound to membrane after DNase treatment (membrane mRNA) and 25% of the kinase mRNA is routinely lost during fractionation. The membrane-bound kinase mRNA is about three times as stable in vitro as the soluble kinase mRNA. Soluble kinase mRNA (14.5S) is found associated with as few as one ribosome and as many as 22 ribosomes; however, 14.5S α-glucosyl transferase mRNA is found predominantly in six ribosome polysomes. The size of the α-glucosyl transferase mRNA is heterogenous, ranging between 14.5 and 20S. The larger α-glucosyl transferase mRNAs are never found on small polysomes but appear only in polysomes containing at least nine ribosomes (18S α-glucosyl transferase mRNA). Maximum-size α-glucosyl transferase mRNA (approximately 20S) appears on polysomes containing at least 14 ribosomes. The relationships between decay of T4 mRNA and polysome size and the location of ribosome loading sites on the 20S α-glucosyl transferase message are also discussed.

T7 protein synthesis in F-factor-containing cells: evidence for an episomally induced impairment of translation and relation to an alteration in membrane permeability.

T7 infection of F-factor-containing PIFA+, B+ cells is abortive. In spite of the presence of mRNA for all three classes of T7 proteins, only the earliest of the T7 proteins are synthesized. A crucial question is whether the failure of T7 to develop in PIFA+, B+ cells is the result of an inability to translate the late classes of T7 mRNA or, as has been recently suggested (Britton, and Haselkorn, 1975; Condit, 1975), whether it is the result of a more generalized alteration in membrane permeability. We have examined the effects of the wild-type PIFA+, B+ spisome and two sipsomal mutations (pifA- and pifB-) on in vitro translation and membrane permeability. In vivo the episomal mutations allow partial or complete T7 development to occur. We demonstrate that cell-free protein-synthesizing systems from T7-infected PIFA+, B+ cells show a three- to fivefold decrease in the rate of translation of both natural and synthetic mRNA. In addition, ribosomes from T7-infected PIFA+, B+ cells are defective in their ability to bind Fmet tRNAf in response to natural mRNA. By contrast, cell-free extracts from T7-infected pifA-(PIFA-, B+) celld retain the ability to bind Fmet defective T7-infected PIFA+, B+ rigosomes can be restored to full activity by a trypsin-sensitive fraction from uninfected PIFA+, B+ or T7-infected PIFA-, B+ cells. Despite the differences in translational capacity of these extracts, both T7-infected PIFA+, B+ and PIFA-, B+ cells display the same permeability lesions as measured by the loss of ATP from the cells into the supernatant. Mutation of the episome of pifB- prevents the loss of ATP from the cells after T7 infection.

The role of IF-3 in the translation of T7-and ϕ80trp messenger RNA

The DNA dependent synthesis of proteins was studied with a system composed of DNA, washed ribosomes, centrifuged (150,000 X g) bacterial extract from Escherichia coli and purified initiation factors IF-1 and IF-2. Synthesis of active enzymes encoded by the tryptophan (trp)-operon of E. coli was found to depend strongly on the addition of IF-3, with the same IF-3 dependency for all 5 gene-products of this operon, irrespective of the presence of the promotor proximal gene trpE. Synthesis of T7 RNA polymerase with T7 DNA as a template, however, was completely independent of the addition of IF-3. The same difference in IF-3 requirement was found when we compared the overall protein synthesis directed by these templates. This difference could be related to the effect of IF-3 on the formation of initiation complexes with the in vitro prepared mRNA: initiation complexes are readily formed with T7 mRNA also in the absence of IF-3, whereas the formation of these complexes with phi80trp mRNA almost completely depends on the presence of this factor.

Analysis of the template activity of bacteriophage T7 messenger RNAs during infection of male and female strains of Escherichia coli

The total template activity and the relative abundance of nine different bacteriophage T7 messenger RNAs has been measured throughout infection of T7-infected male and female cells by measuring quantitatively the amount of gene-specific polypeptide synthesized in S-30 extracts prepared from uninfected Escherichia coli cells. The total template activity of RNA extracted from two T7-infected male strains, E. coli 279/F′ and E. cli RV/F′, is 20% and 50%, respectively, of the template activity in RNA extracted from T7-infected, isogenic female strains. At the individual mRNA level, there is not a uniform reduction in the level of the nine different messenger RNAs assayed. The early messages for T7 RNA polymerase and the gene 0.3 protein are present in equal amounts at early times in infected male and female cells. At late times there is a higher apparent concentration of 0.3 mRNA in male cells. Among the late proteins there is a wide variation in relative mRNA abundance in male cells. The mRNA for T7 lysozyme is present in equal amounts in male and female cells. The late mRNAs encoded by genes 6, DUP, 8, 9, 10 and 15 are present in RNA extracted from male cells at concentrations ranging from 5 to 100% of their level in female cells. The messenger RNAs corresponding to genes 16, 17 and 19 have not been detected in RNA from male cells although they can be detected in RNA from female cells. The mRNA from gene 12 is present in RNA from male cells but at a concentration too low to measure accurately. The relative amount of individual T7 mRNAs is lower in E. coli 279/F′ than it is in RV/F′, but there is a qualitative consistency in the degree of reduction in the two male strains. When S-30 extracts of T7-infected male and female cells are compared for their ability to translate added T7 early or late mRNA, there is an insignificant difference between the two extracts. Both are able to translate late T7 mRNAs with approximately equal efficiency. This efficiency is similar to the efficiency of translation in an S-30 extract prepared from uninfected male or female cells. Thus it appears that there is no specific translational discrimination against late T7 mRNAs in infected or uninfected male E. coli cell extracts. The unknown cause of the abortive T7 infection of male cells appears to reduce the synthesis of certain late T7 messenger RNAs as well as reducing total late protein synthesis.

The effect of tRNA derivatives bound with natural or synthetic mRNA on the interaction of Escherichia coli ribosomes with colicin E3.

Ribosomal binding complexes directed by poly(U) or T4 mRNA were formed with aminoacyl-tRNA or its derivatives bound to predominantly the P or A binding site. The defined binding complexes were reacted with colicin E3 and the reaction was assessed by the ability of the complexes to proceed with polypeptide synthesis. The results indicated that only one of the four complexes tested was completely resistant to colicin E3-induced inactivation: that of Phe-tRNA bound in the presence of poly(U) to the A-site. The poly(U) directed complex of AcPhe-tRNA and the T4-mRNA-directed complex at the A-site appeared slightly resistant, while the T4 mRNA initiation complex was inactivated by colicin E3 in a manner similar to non-complexed ribosomes. Colicin E3 added to ribosomes after protein synthesis had been initiated affected the subsequent polymerization in a manner corresponding to the response of the binding complexes. Thus, poly(U)-translating ribosomes were less affected than ribosomes translating the viral mRNA. The vulnerability of natural-mRNA-directed binding complexes to inactivation by colicin E3 is in accord with the mode of inactivation by the colicin in vivo.

The host factor required for RNA phage Qbeta RNA replication in vitro. Intracellular location, quantitation, and purification by polyadenylate-cellulose chromatography.

The Qbeta host factor, a heat-stable protein necessary in concert with Qbeta replicase for phage Qbeta RNA replication in vitro, has been localized in Escherichia coli and found to be associated primarily with ribosomes. This location has been established both by complement fixation assays with highly specific antiserum directed against the host factor, and by in vitro stimulation of Qbeta RNA replication by the Qbeta replicase. The complement fixation assay has provided the estimate that there are approximately 2500 copies of the host factor polypeptide per cell. The host factor is released from the ribosomes by a 1 M NH4Cl wash and concentrated by ammonium sulfate precipitation. It can be purified to apparent homogeneity in one further step by chromatography on poly(A)-cellulose. Ribosomal protein S1 subunit I of Qbeta replicase) also binds to the poly(A)-cellulose column and elutes before the host factor. In agreement with previous reports, we find that the host factor has a monomer molecular weight of 12,000 as judged by sodium dodecyl sulfate-polyacrylamide gels, and a native molecular weight of 72,000 as judged by the stoichiometric interaction of the host factor with Qbeta RNA, by sedimentation in sucrose velocity gradients, and by sodium dodecyl sulfate gel mobility when incompletely disaggregated. The Qbeta host factor is a potent inhibitor of an in vitro poly(A)-directed polylysine protein-synthesizing system, but has less effect on the in vitro translation of poly(U), R17 RNA, late T7 mRNA, or endogenous E. coli mRNA. The amino acid composition and NH2- terminal sequence rule out the host factor as one of the known 30 S or 50 S E. coli ribosomal proteins. The finding that the Qbeta host factor is associated with ribosomes in vivo completes the demonstration that all of the host-supplied proteins required for phage Qbeta RNA replication in vitro are either associated with ribosomes or are involved in the protein-synthetic machinery of the cell.