Localization of translation initiation in the message for E. coli UDP-galactose 4-epimerase: the amino terminal sequence

The amino acid sequence of $\text{E.}$$\text{coli}$ UDP-galactose 4-epimerase has been determined through the amino-terminal 28-amino acid residues using an automated protein sequenator. Alignment of UDP-galactose operon messenger RNA and the amino acid sequence of epimerase demonstrates that the first 26 bases in the mRNA are transcribed but do not take part in translation of epimerase.     

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 $\text{in vivo}$.     

Endonuclease of high specific activity in a purified mouse interferon preparation

We find an endonuclease of high specific activity in a purified mouse interferon preparation. The interferon was purified from Ehrlich ascites tumor cultures which were induced with Newcastle disease virus. It has a higher specific activity (1.5 × 10⁹ NIH mouse reference standard interferon units/mg protein) than reported for any interferon preparation but is not homogeneous. We do not know if the endonuclease activity is due to a contaminating protein or to interferon. The endonuclease does not degrade in our conditions polyuridylic acid or double stranded reovirus RNA and does not inactivate the tRNA₂^Gln species from $\text{E. coli}$, or tRNA^Val species or polysomes from mouse L cells. Endonuclease in as little as 0.5 ng protein of the interferon preparation degrades μg quantities of messenger RNA from mouse L cells, of encephalomyocarditis virus RNA and of $\text{in vitro}$-synthesized reo-virus messenger RNA at 37° in 1 hour. Further characteristics of the endonuclease and its possible relationship (if any) to interferon remain to be established.     

The in vivo equivalence of a cell-free system for RNA processing and transport

The equivalence of messenger RNA released (transported) from isolated rat liver nuclei to three selected media, with messenger RNA normally released to liver cytoplasm $\text{in vivo}$, has been evaluated by competitive DNA: RNA hybridization. Near normal nuclear restriction was exhibited by nuclei in media fortified with ATP, salts, spermidine and dialyzed cytosol. The RNA transport in the latter system was markedly inhibited by colchicine as was also the transport of RNA $\text{in vivo}$. Both nuclear restriction and sensitivity of the RNA transport to colchicine in media lacking spermidine and cytosol deviated significantly from the $\text{in vivo}$ norm. The results emphasize the importance of establishing the $\text{in vivo}$ equivalence in cell-free systems designed to study RNA synthesis, processing and transport.     

Competition between bacteriophage f2 RNA and bacteriophage T4 messenger RNA

In an $\text{Escherichia coli}$ cell-free protein synthesis assay, mRNA isolated from cells late after infection by phage T4 out-competes bacteriophage f2 RNA. Addition of a saturating or subsaturating amount of T4 mRNA inhibits translation of f2 RNA, while even an excess of f2 RNA has no effect on translation of T4 mRNA. Peptide mapping of reaction products labeled with formyl-[³⁵S]-methionyl-tRNA was used to quantitate f2 and T4 protein products synthesized in the same reaction. We suggest that messenger RNA competition might be one mechanism by which T4 superinfection of cells infected with phage f2 blocks translation of f2 RNA and possibly host mRNA.     

The early synthesis and possible function of a 0.5 X 10(6) Mr RNA after transfer of dark-grown Spirodela plants to light.

A 0.5 × 10⁶$\text{M}$_r RNA found in plastids of the aquatic angiosperm $\text{Spirodela}$, is synthesized at a much higher rate than any other rapidly labeling RNA species about $\text{3–3}\text{1}\text{2}$ h after dark-grown plants are transferred to light. The pulse labeling kinetics of the 0.5 × 10⁶$\text{M}$_r RNA after transfer to light, argue against its involvement in the biogenesis of plant rRNAs. Although poly(A) RNA is found in $\text{Spirodela}$, poly(A) sequences are not detected in the 0.5 × 10⁶$\text{M}$_r RNA; yet a sucrose gradient fraction which includes RNA of this $\text{M}$_r stimulates amino acid incorporation by an $\text{E. coli}$ cell free extract more than other RNA fractions. The possible involvement of the 0.5 × 10⁶$\text{M}$_r RNA as a chloroplast messenger is discussed.     

Inefficiency of the stringent response in the fungus Mucor

Removal of a required amino acid from the growth medium or addition of cycloheximide caused an immediate stoppage of growth and protein synthesis in the fungus $\text{Mucor}\text{racemosus}$. However, RNA synthesis persisted for several hours at rates that only gradually decreased under the same circumstances. An analysis of the major classes of RNA synthesized during the first hour of treatment showed that cycloheximide preferentially inhibited rRNA synthesis, whereas amino acid starvation slowed synthesis of all RNA species uniformly. Neither treatment affected the percentage of mRNA synthesized. The partial and delayed effects of amino acid starvation and cycloheximide treatment on RNA synthesis reported here suggest the absence of or the gross inefficiency of a classical stringent response in $\text{M.}\text{racemosus}$.     

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

Effect of 5-fluoroorotic acid administration of the 32 P base composition, DNA-RNA hybridization properties, and labeling of polyadenylate-rich RNA in the cytoplasm of rat liver cells

5-Fluoroorotic acid treatment lowered the (Guanine + Cytosine)/(Adenine + Uracil) base ratio of ³²P-labeled microsomal RNA from a control value of 1.36 to 1.00. Low doses of actinomycin D, which are effective in inhibiting ribosomal RNA synthesis without significantly affecting messenger RNA synthesis, caused a similar decrease in the base ratio. Microsomal RNA labeled by [³H]orotate in the presence of 5-fluoroorotic acid had approximately $\text{1}\text{2}$ the specific radioactivity but twice the hybridization efficiency of RNA labeled in its absence. Evidence is presented that this RNA (1) has a different structure from that of ribosomal RNA, (2) hybridizes to DNA with an efficiency consistent with that of other published studies of polysome-associated messenger RNA, and (3) possesses sequences which are present in other samples of liver microsomal RNA but not in kidney microsomal RNA. These properties differ from those known to be exhibited by 18 S and 28 S ribosomal RNA. Electrophoretic analysis of this [³H]orotate-labeled microsomal RNA indicated that the analogue greatly inhibited precursor incorporation into ribosomal RNA but had little or no effect on incorporation into messenger RNA. Ribosomal RNA and polyadenylate-rich nonribosomal RNA were prepared from total polyribosomes by phenol extraction at pH 7.6 and pH 9.0, respectively. 5-Fluoroorotic acid inhibited [³H]orotate or ³²P_i incorporation into the pH 7.6 fraction much more effectively than incorporation into the pH 9.0 fraction. A subfraction of the pH 9.0 RNA which was retained by a polythymidylate-cellulose column had a greatly increased adenylate content.     

The effect of dietary fat on the activity,content, rates of synthesis,and degradation and translation of messenger RNA coding for malic enzyme in mouse liver

The specific activity (units activity/mg cytosolic protein) of malic enzyme was found to be three-fold higher in the livers of mice fed a semipurified diet containing 50% ($\text{w}\text{w}$) glucose and 15% ($\text{w}\text{w}$) saturated and monounsaturated but no polyunsaturated fat (hydrogenated cottonseed oil) over an 11-day period than in the livers of mice fed a standard laboratory mouse chow (Purina) diet. In contrast, when other lab chow-fed mice were fed an isocaloric diet containing 15% ($\text{w}\text{w}$) polyunsaturated fat (corn oil), no change in the specific activity of malic enzyme occurred over a similar period of time. Rocket immunoelectrophoresis performed on cytosols from both dietary groups demonstrated that the livers of mice consuming the hydrogenated cottonseed oil diet contained approximately three times more malic enzyme protein than did the livers from the corn oil-fed animals. In mice pulse-labeled with l-[4,5-³H]leucine, the rate of hepatic malic enzyme synthesis (relative to that for total protein) was approximately twofold greater in the hydrogenated cottonseed oil-fed mice than in their corn oil-fed counterparts whereas the rate of hepatic malic enzyme degradation was similar for both groups. Immunotitration of liver malic enzyme from hydrogenated cottonseed oil-fed and corn oil-fed mice revealed identical equivalence points, demonstrating that the catalytic efficiency of mouse liver malic enzyme had not been affected by the type of dietary fat administered. When total liver RNA, isolated from the hydrogenated cottonseed oil- and the corn oil-fed animals, was translated in cell-free translation systems (wheat germ extract and reticulocyte lysate) we found that both dietary treatments had resulted in an increase in the activity of malic enzyme messenger RNA. Furthermore, there were no significant differences between the two dietary groups in this regard. These results suggest that hepatic malic enzyme specific activity in high-carbohydrate polyunsaturated fat-fed mice is regulated principally by dietary-induced changes in the rate of enzyme synthesis and not by the activity of messenger RNA coding for the enzyme.     

Effect of ammonia and glutamine on macromolecule synthesis and breakdown during sporulation of Saccharomyces cerevisiae.

The effect of two known inhibitors of sporulation in yeast, ammonia and glutamine, on certain biochemical events during sporogenesis have been studied using sporulating $\text{a}\text{α}$ and non sporulating $\text{α}\text{α}$ cells. Both strains gave similar results on the increase in dry cell weight, protein and RNA breakdown and the suppression of the intensive RNA and protein syntheses occurring after 4 hours. The inhibitory effect of ammonia and glutamine on RNA and protein syntheses is reversible under the same conditions which do so for sporulation.     

Histone mRNA in eggs and embryos of Strongylocentrotus purpuratus

Histone messenger RNA is detectable in both the maternal RNA which is stored in the unfertilized sea urchin egg and in the RNA species which are synthesized $\text{de}$$\text{novo}$ after fertilization. Hybridization competition experiments show that sequences similar to pulse-labeled 912S RNA from morulae are present in total RNA from unfertilized eggs as well as that from later stages. The proportion of histone mRNA in cellular RNA increases after fertilization, reaching a maximum at the morula stage. Although these messengers are still present in hatched blastulae and gastrulae, they represent a smaller proportion of total RNA compared with earlier stages.     

RNA-protein interactions in the ribosome. I. Characterization and ribonuclease digestion of 16 S RNA-ribosomal protein complexes

Proteins from the 30 S ribosomal subunit of Escherichia coli were fractionated by column chromatography and individually incubated with 16 S ribosomal RNA. Stable and specific complexes were formed between proteins S4, S7, S8, S15 and S20, and the 16 S RNA. Protein S13 and one or both proteins of the $\text{S}\text{16}\text{S}\text{17}$ mixture bound more weakly to the RNA, although these interactions too were apparently specific. The binding of $\text{S}\text{16}\text{S}\text{17}$ was found to be markedly stimulated by proteins S4, S8, S15 and S20. Limited digestion of the RNA-protein complexes with T₁ or pancreatic ribonucleases yielded a variety of partially overlapping RNA fragments, which retained one or more of the proteins. Since similar fragments were recovered when 16 S RNA alone was digested under the same conditions, their stability could not be accounted for by the presence of bound protein. The integrity of the fragments was, however, strongly influenced by the magnesium ion concentration at which ribonuclease digestion was carried out. Each of the RNA fragments was characterized by fingerprinting and positioned within the sequence of the 1600-nucleotide 16 S RNA molecule. The location of ribosomal protein binding sites was delimited by the pattern of fragments to which a given protein bound. The binding sites for proteins S4, S8, S15, S20 and, possibly, S13 and $\text{S}\text{16}\text{S}\text{17}$ as well, lie within the 5′-terminal half of the 16 S RNA molecule. In particular, the S4 binding site was localized to the first 500 nucleotides of this sequence while that for S15 lies within a 140-nucleotide sequence starting about 600 nucleotides from the 5′-terminus. The binding site for the protein S7 lies between 900 and 1500 nucleotides from the 5′-terminus of the ribosomal RNA.