Donald P. Nierlich

Radioisotope Uptake as a Measure of Synthesis of Messenger RNA

Exogenously supplied radioactive uracil (or guanine) enters the intracellular pools of RNA precursors in Escherichia coli only as nucleotides are removed from these pools by net synthesis of RNA. Consequently uptake of uracil over a short period does not measure the sum of the synthesis of all forms of RNA, unstable and stable, as is often supposed. Uptake of uracil during changing conditions of growth may be influenced by changes in types of RNAs being made; under such conditions that no stable RNA is being made, the synthesis of unstable forms may be greatly underestimated.

Regulation of ribonucleic acid synthesis in growing bacterial cells: I. Control over the total rate of RNA synthesis

Abstract It is well known that bacterial cells regulate their rate of RNA accumulation (net RNA synthesis) in strict accord with the potential of a particular medium to support growth at a given rate, and that in shifts from one medium to another there are rapid and seemingly preferential changes in the net rate, while the other parameters of cell growth change more slowly. In this study, in addition to making measurements of net rates of RNA synthesis in steady states and shifts from one medium to another, measurements of the total rate, that is the sum of the rates of synthesis of stable and unstable RNA species, are made. This is done by measuring the early slope of the incorporation of a radioactive precursor ([3H]guanine) into RNA, and correcting for the specific activity of the intracellular precursor ([3H]GTP) at those times. It is found that, in differing steady states of growth, cells vary, not only their total rate of RNA synthesis, but also the fraction of the total given to the formation of unstable species. Expressed as a ratio of stable to unstable RNAs, this latter value is about one to one in minimal-glucose and two to one in medium enriched with amino acids. Moreover, transients between steady states give a stronger indication that the synthesis of stable and unstable RNA species can vary independently. In shift-up for example, while the net rate of accumulation, presumably largely the synthesis of the stable ribosomal and transfer RNAs, increases immediately, giving rise to the striking increase of isotope uptake known to take place, the rate of total synthesis increases only in the course of 5 to 15 minutes, such that at early times the ratio of stable to unstable RNAs made reaches about three to one. These experiments suggest that the mechanism for the control of RNA synthesis involves both a means whereby the total capacity of the cell to make RNA is limited, as well as a mechanism by which the distribution of that capacity between template sites leading to the synthesis of stable and unstable RNA species is regulated.

Regulation of bacterial growth.

Is the control of bacterial metabolism so complex? The answer can be found in a simple experiment. Two cultures of bacteria are grown in different mediums. One contains as the carbon and nitrogen sources a mixture of amino acids, while the other contains only glucose and ammonia, so that the cells must synthesize all of the amino acids. The results show that insofar as the cells in both cultures grow at comparable rates, they will have the same composition in terms of DNA, RNA, and protein (30). To explain this phenomena I have argued that through the control mechanisms responsible for the distribution of substrates in intermediary metabolism, the substrates of protein synthesis are produced at concentrations and rates commensurate with the ability of the environment to support growth. The provision of these substrates relative to the ability of the protein forming system to utilize them regulates the synthesis of ribosomal and transfer RNA, which, after adjustment for various modulating influences, such as nonfunctioning ribosomes or ribosomal RNA turnover, brings the number of functioning ribosomes to a point in keeping with the provision of external nutrients. The synthesis of messenger (or total) RNA, ribosomal proteins, and DNA, and the process of cell division, for example, are subject to their own controls, but through the burden they each place on intermediary metabolism, they provide a means for partitioning the cells metabolic resources. It might be noted that this view may not be very far from the idea once held that the rate at which each of the transfer RNAs was changed by amino acids regulate the synthesis of bacterial RNA, but growth regulation is clearly more complicated than implied by that model (76).

Regulation of ribonucleic acid synthesis in growing bacterial cells: II. Control over the composition of the newly made RNA☆

Abstract Studies have been made by base-ratio analysis and DNA-RNA hybridization of the pulse-labeled, nascent RNA in cells of Escherichia coli growing exponentially in minimal-glucose and Casamino acids-supplemented media, as well as during shift-ups or shift-downs between them. It is found that the cell varies the fraction of the newly made RNA given over to the synthesis of ribosomal and (presumably) transfer RNA under these different conditions. Furthermore, for cells in exponential growth or during shift-up, these values are in good agreement with the amount of “stable” RNA made as defined in the kinetic experiments in the accompanying paper. Thus under these conditions, there is no need to postulate the existence of a mechanism involving the rapid destruction of otherwise stable RNAs, a situation which may occur in shift-down or other growth conditions. During shifts, the synthesis of messenger may transiently fall (shift-up), while protein synthesis continues, or the synthesis of messenger may continue (shift-down), while protein synthesis is restricted. These and other experiments suggest that the cell has a means of regulating the translation of messenger generally, and that no obligate coupling exists between the synthesis of messenger and its translation.

Functional Characterization of the Dimer Linkage Structure RNA of Moloney Murine Sarcoma Virus

ABSTRACT Several determinants that appear to promote the dimerization of murine retroviral genomic RNA have been identified. The interaction between these determinants has not been extensively examined. Previously, we proposed that dimerization of the Moloney murine sarcoma virus genomic RNAs relies upon the concentration-dependent interactions of a conserved palindrome that is initiated by separate G-rich stretches (H. Ly, D. P. Nierlich, J. C. Olsen, and A. H. Kaplan, J. Virol. 73:7255–7261, 1999). The cooperative action of these two elements was examined using a combination of genetic and antisense approaches. Dimerization of RNA molecules carrying both the palindrome and G-rich sequences was completely inhibited by an oligonucleotide complementary to the palindrome; molecules lacking the palindrome could not dimerize in the presence of oligomers that hybridize to two G-rich sequences. The results of spontaneous dimerization experiments also demonstrated that RNA molecules lacking either of the two stretches of guanines dimerized much more slowly than the full-length molecule which includes the dimer linkage structure (DLS). However, the addition of an oligonucleotide complementary to the remaining stretch of guanines restored the kinetics of dimerization to wild-type levels. The ability of this oligomer to rescue the kinetics of dimerization was dependent on the presence of the palindrome, suggesting that interactions within the G-rich regions produce changes in the palindrome that allow dimerization to proceed with maximum efficiency. Further, unsuccessful attempts to produce heterodimers between constructs lacking various combinations of these elements indicate that the G-rich regions and the palindrome do not interact directly. Finally, we demonstrate that both of these elements are important in maintaining efficient viral replication. Modified antisense oligonucleotides targeting the DLS were found to reduce the level of viral vector titer production. The reduction in viral titer is due to a decrease in the efficiency of viral genomic RNA encapsidation. Overall, our data support a dynamic model of retroviral RNA dimerization in which discrete dimerization elements act in a concerted fashion.

Ribosomal subunit entry into polysomes in yeast

The kinetics of entry of newly synthesized 40 S and 60 S ribosomal subunits into yeast polysomes is described. The entry times for 40 S and 60 S subunits were found to be 3 and 8 min, respectively. The kinetics of entry of 40 S subunits into large polysomes is found to be different from the kinetics of entry of 60 S subunits into large polysomes.

The methylation of transfer RNA in Escherichia coli

Abstract Analysis of both RNA pulse-labeled in vivo with L-[Me-3H]methionine and pools of methyl-accepting RNA from normally growing Escherichia coli indicates that methylation, by and large, takes place on molecules of the same size as mature tRNA rather than on longer precursor molecules. Two forms of such methyl-accepting tRNAs have been detected. One form, co-fractionating on methylated albumin—kieselguhr columns with mature tRNA, is apparently similar in structure to mature tRNA. The other form, fractionating similarly on methylated albumin columns to transfer RNA molecules lacking the modified nucleoside pseudouridine (prepared by substituting 5-fluorouracil for uracil in tRNA), apparently lacks this modified nucleoside. Using model substrates similar to the cellular substrates described above, kinetic studies were performed both in vitro and in vivo to determine whether there is a random or an ordered formation of the individual methylated bases in E. coli tRNA. Results indicate that, with the possible exception of 2-methyladenine formation, E. coli tRNA methylating enzymes act in random sequence in catalyzing the methylation of tRNA.

Use of the lac repressor in constructing sequencial deletions and a new sequencing vector

Large sequencing projects require an efficient strategy to generate a series of overlapping clones. This can be accomplished by protecting one end of a linear DNA molecule while sequential deletions are introduced into the other end by exonuclease digestion. We demonstrate that the lac repressor can protect the ends of linear nucleotide sequences from digestion by exonuclease if these ends contain the lac operator sequence. To exploit this, we have inserted the lac operator sequence between the primer-binding site and multiple cloning site of an M13 sequencing vector. Linearizing the replicative form and binding lac repressor protein protects the end next to the vector sequences. Sequential deletions are then introduced into the insert by digesting with exonuclease III or BAL 31. Because the rate and time of digestion are readily controlled, the region brought next to the sequencing primer site, after religation, can be selected in a timed series of reactions. This minimizes the screening needed to isolate an overlapping series of clones and facilitates sequencing of long regions.

Yeast mutant, rna1, affects the entry into polysomes of ribosomal RNA as well as messenger RNA

SummaryThe entry of newly labeled ribosomal subunits and mRNA into polysomes was examined in the yeast mutant rna1. The entry of both types of RNA into polysomes is inhibited rapidly at the restrictive temperature. Analysis of the labeling of the ATP pool and the kinetics of synthesis and processing of mRNA at the restrictive temperature leads to the conclusion that the primary defect in the mutant affects transport of both ribosomes and messenger across the nuclear membrane.

Isolation of the transfer RNA genes of bacteriophage T4 and transfer RNA synthesis in vitro

Non-glucosylated T4 DNA was restricted with the endonuclease EcoRI and the mixture of DNA fragments separated by gel electrophoresis and transcribed with purified Escherichia coli RNA polymerase. Three purified fragments were shown to act as templates for tRNA synthesis. A smaller fragment, shown to be hybridizable to 32P-labeled T4 tRNA was not transcribable. It was concluded that the promoter for T4 tRNA synthesis had been separated from the structural genes in the smaller fragment by EcoRI and that the distal portion of the tRNA gene cluster lacks internal promoters which display in vitro activity. Preparations of non-glucosylated T4 DNA were never fully restricted with EcoRI and when the larger purified fragments carrying the tRNA were restricted with excess enzyme only a slight cleavage to yield the smaller fragments was obtained. The property of the DNA-limiting complete restriction is not know.