The role of ribosomes in stabilizing bacteriophage T4 deoxynucleotide kinase mRNA.

In the present investigation, an approach toward defining the role of ribosomes in stabilizing functional messenger RNA in cell-free extracts is described. The data presented show that initiation of protein synthesis is necessary for maximal functional stability of bacteriophage T4 deoxynucleotide kinase mRNA $\text{in vitro}$ and suggest that much of the stability is attained by interaction of the deoxynucleotide kinase mRNA initiation site with a 30S ribosomal subunit. Data is also presented which suggest that any of several $\text{E}$. $\text{coli}$ ribonucleases could serve as a messenger ribonuclease $\text{in vivo}$.     

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).     

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.     

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.     

The virion host shutoff protein of herpes simplex virus type 1: messenger ribonucleolytic activity in vitro.

Shortly after tissue culture cells are infected with herpes simplex virus (HSV) type 1 or 2, the rate of host protein synthesis decreases 5- to 10-fold and most host mRNAs are degraded. mRNA destabilization is triggered by the virion host shutoff (vhs) protein, a virus encoded, 58-kDa protein located in the virion tegument. To determine whether it can function as a messenger RNase (mRNase), the capacity of vhs protein to degrade RNA in vitro in absence of host cell components was assessed. Two sources of vhs protein were used in these assays: crude extract from virions or protein translated in a reticulocyte-free system. In each case, wild-type but not mutant vhs protein degraded various RNA substrates. Preincubation with anti-vhs antibody blocked RNase activity. These studies do not prove that vhs protein on its own is an mRNase but do demonstrate that the protein, either on its own or in conjunction with another factor(s), has the biochemical property of an mRNase, consistent with its role in infected cells.     

Specificity of protein synthesis by bacterial ribosomes and initiation factors: Absence of change after phage T4 infection

Using several natural messenger RNA's—f2 RNA, Qβ RNA, T7 RNA, T4 early mRNA, T4 late mRNA and Escherichia coli RNA—ribosomes isolated from cells either 5 or 12 minutes after T4 infection direct synthesis of only 35 to 70% as much protein as do ribosomes from uninfected cells. However, with poly(U) or formaldehyde-treated f2 RNA message, both types of ribosomes work equally well. Experiments mixing salt-washed ribosomes and initiation factors from these cells show, in agreement with work of others, that the reduction with natural messages is due only to changes in the initiation factors.     

4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle

Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesis-degradation equilibrium that is conditionally modulated under an extensive range of physiological and pathophysiological circumstances. Recent studies have established the initiation phase of mRNA translation as a pivotal site of regulation for global rates of protein synthesis, as well as a site through which the synthesis of specific proteins is controlled. The protein synthetic pathway is exquisitely sensitive to the availability of hormones and nutrients and employs a comprehensive integrative strategy to interpret the information provided by hormonal and nutritional cues. The translational repressor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1) have emerged as important components of this strategy, and together they coordinate the behavior of both eukaryotic initiation factors and the ribosome. This review discusses the role of 4E-BP1 and S6K1 in translational control and outlines the mechanisms through which hormones and nutrients effect changes in mRNA translation through the influence of these translational effectors.     

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.     

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 μm, 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.     

Nucleotide sequence from the genetic left end of bacteriophage T7 DNA to the beginning of gene 4

The nucleotide sequence running from the genetic left end of bacteriophage T7 DNA to within the coding sequence of gene 4 is given, except for the internal coding sequence for the gene 1 protein, which has been determined elsewhere. The sequence presented contains nucleotides 1 to 3342 and 5654 to 12,100 of the approximately 40,000 base-pairs of T7 DNA. This sequence includes: the three strong early promoters and the termination site for Escherichia coli RNA polymerase: eight promoter sites for T7 RNA polymerase; six RNAase III cleavage sites; the primary origin of replication of T7 DNA; the complete coding sequences for 13 previously known T7 proteins, including the anti-restriction protein, protein kinase, DNA ligase, the gene 2 inhibitor of E. coli RNA polymerase, single-strand DNA binding protein, the gene 3 endonuclease, and lysozyme (which is actually an N-acetylmuramyl-l-alanine amidase); the complete coding sequences for eight potential new T7-coded proteins; and two apparently independent initiation sites that produce overlapping polypeptide chains of gene 4 primase. More than 86% of the first 12,100 base-pairs of T7 DNA appear to be devoted to specifying amino acid sequences for T7 proteins, and the arrangement of coding sequences and other genetic elements is very efficient. There is little overlap between coding sequences for different proteins, but junctions between adjacent coding sequences are typically close, the termination codon for one protein often overlapping the initiation codon for the next. For almost half of the potential T7 proteins, the sequence in the messenger RNA that can interact with 16 S ribosomal RNA in initiation of protein synthesis is part of the coding sequence for the preceding protein. The longest non-coding region, about 900 base-pairs, is at the left end of the DNA. The right half of this region contains the strong early promoters for E. coli RNA polymerase and the first RNAase III cleavage site. The left end contains the terminal repetition (nucleotides 1 to 160), followed by a striking array of repeated sequences (nucleotides 175 to 340) that might have some role in packaging the DNA into phage particles, and an A · T-rich region (nucleotides 356 to 492) that contains a promoter for T7 RNA polymerase, and which might function as a replication origin.     

Effect of T4 infection on initiation of protein synthesis and messenger specificity of initiation factor 3

No alteration in the messenger specificity of initiation factor 3 (IF-3) is observed upon T4 phage infection of several strains of Escherichia coli. IF-3 present in the 1.0 m NH₄Cl washes of ribosomes from T4-infected cells supports the translation of f2 RNA and T4 late mRNA with the same degree of efficiency as the IF-3 in the ribosomal washes obtained from uninfected cells. At high concentrations the ribosomal washes obtained from T4-infected cells are more inhibitory for both f2 RNA- and T4 late mRNA-directed protein synthesis than the ribosomal washes from uninfected cells. Furthermore, this increased inhibition is also observed in the poly(U)-directed synthesis of polyphenylalanine. These data suggest that translational controls exerted at the level of IF-3 probably do not account for the alterations in protein synthesis observed upon T4 infection.     

High-throughput assays probing protein-RNA interactions of eukaryotic translation initiation factors

Protein-RNA interactions are involved in all facets of RNA biology. The identification of small molecules that selectively block such bimolecular interactions could provide insight into previously unexplored steps of gene regulation. Such is the case for regulation of eukaryotic protein synthesis where interactions between messenger RNA (mRNA) and several eukaryotic initiation factors govern the recruitment of 40S ribosomes (and associated factors) to mRNA templates during the initiation phase. We have designed simple fluorescence polarization-based high-throughput screening assays that query the binding of several translation factors to RNA and found that the mixed inhibitor p-chloromercuribenzoate interferes with poly(A) binding protein-RNA interaction.     

In polycistronic Q�� RNA, single-strandedness at one ribosome binding site directly affects translational initiations at a distal upstream cistron

In Qβ RNA, sequestering the coat gene ribosome binding site in a putatively strong hairpin stem structure eliminated synthesis of coat protein and activated protein synthesis from the much weaker maturation gene initiation site, located 1300 nucleotides upstream. As the stability of a hairpin stem comprising the coat gene Shine–Dalgarno site was incrementally increased, there was a corresponding increase in translation of maturation protein. The effect of the downstream coat gene ribosome binding sequence on maturation gene expression appeared to have occurred only in cis and did not require an AUG start codon or initiation of coat protein synthesis. In all cases, no structural reorganization was predicted to occur within Qβ RNA. Our results suggest that protein synthesis from a relatively weak translational initiation site is greatly influenced by the presence or absence of a stronger ribosome binding site located elsewhere on the same RNA molecule. The data are consistent with a mechanism in which multiple ribosome binding sites compete in cis for translational initiations as a means of regulating protein synthesis on a polycistronic messenger RNA.