Ribonuclease F,a putative processing endoribonuclease from Escherichia coli

A new endoribonuclease activity, RNase F, was partially purified from $\text{Escherichia coli}$ cells. This activity can cleave a precursor RNA molecule (of Species 1), isolated from T4 infected cells, in a specific site. This activity is different from the other three know processing endoribonucleases of $\text{E. coli}$ RNase III, RNase E and RNase P.     

Phosphorylated guanosine derivatives of eukaryotes: Regulation of DNA-dependent RNA polymerases I,II, and III in fungal development

Three phosphorylated guanosine derivatives designated HS-1, HS-2 and HS-3 synthesised during active protein synthesis in the water-mould, $\text{Achlya}$ sp (1969) were shown to regulate the enzymatic activities of nucleoplasmic and nucleolar DNA-dependent RNA polymerases (RNAP-I, II and III) from both $\text{Achlya}$ and another unrelated water-mould, $\text{Blastocladiella emersonii}$. These HS compounds were without effect on $\text{E. coli}$ DNA-dependent RNA polymerase holoenzyme. The most potent of the three compounds was HS-3 which inhibited the activity of all enzymes completely at 100 μg/ml. HS-1, on the other hand, activated maximally at 1 to 10 μg/ml. HS-1 activation (3-fold) was restricted to enzyme III, and it had only partial inhibitory effects on enzymes I and II. The pattern of synthesis of HS-compounds throughout the 20-hour asexual growth cycle of the organism correlated with the detectable levels of the different RNA polymerases of $\text{Achlya}$.     

A possible complex containing RNA processing enzymes

The three enzymes, RNAase III, RNAase E and RNAase P participate in the processing of RNA precursors in $\text{Escherichia coli}$. In extracts which contain a mutated RNAase III or RNAase E under certain conditions RNAase P activity is not expressed while in the wild-type extract it is. Upon high-speed centrifugation of a cell extract from a strain of $\text{E.}$,$\text{coli}$, which contains all these three enzymes, the majority of RNAase P, RNAase III and RNAase E activities sediment as particles heavier than their known sizes. In a sucrose density gradient of the cell extract, part of RNAase E and RNAase P activities co-sediment while most of the RNAase III activity is found toward the top of the gradient. This behavior is distinct from other ribonucleases such as RNAase II and RNAase H, which do not sediment as complexes. This complex does not seem to be caused merely by the association of the enzymes with ribosomes.     

Two routes for synthesis of phosphoenolpyruvate from C4-dicarboxylic acids in Escherichia coli

Mutants of $\text{E. coli}$ defective in both phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthetase are unable to use C₄-dicarboxylic acids such as succinate and malate as carbon and energy sources for growth. Revertants that have restored function for either one of these enzymes can grow in a malate-mineral medium, but at a reduced rate compared with the growth of wild-type cells. $\text{E. coli}$ appears to use two pathways for synthesis of phosphoenolpyruvate from C₄-dicarboxylic acids. One of these involves decarboxylation of oxalacetate catalyzed by phosphoenolpyruvate carboxykinase. The second pathway makes use of the combined action of malic enzyme and phosphoenolpyruvate synthetase.     

Studies on invitro DNA synthesis: II. Isolation of a protein which stimulates deoxynucleotide incorporation catalyzed by DNA polymerases of E.coli

Purified RNA polymerase, DNA polymerase III and unwinding protein of $\text{Escherichia}\text{coli}$ catalyze limited rifampicin sensitive fd or ØX 174 DNA-dependent DNA synthesis. A protein has been partially purified from $\text{E.}\text{coli}$ which stimulates rifampicin sensitive dXMP incorporation in this system 20 to 30 fold. This protein also stimulates DNA synthesis catalyzed by DNA polymerases I and II; the stimulation occurs in reactions primed with natural and synthetic DNAs as well as RNA-DNA hybrids. The protein is not a product of the known dna genes. In contrast to the above system of purified enzymes, rifampicin sensitive dXMP incorporation in crude extracts of $\text{E.}\text{coli}$ is specifically dependent on fd but not ØX 174 DNA. An additional factor has been isolated from extracts of $\text{E.}\text{coli}$ which restores specificity to the purified rifampicin sensitive system by preventing ØX 174 DNA from serving as a template.     

Rifamycin derivatives: specific inhibitors of nucleic acid polymerases

Rifampicin and three rifamycin SV derivatives with different lipophilic side chains were tested as inhibitors of a number of purified enzymes including the α and αβ forms of RNA-directed DNA polymerase of avian myeloblastosis virus (AMV). $\text{AF}\text{ABDMP}$ (2,5-dimethyl-4-N-benzyl demethyl rifampicin), $\text{AF}\text{013}$ (O-n-octyloxime of 3-formyl rifamycin SV) and C-27 (rifamycin SV with a dicyclohexylalkyl substituted piperidyl ring at the 3-position) at concentrations less than 20 to 40 μg/ml completely inhibited the RNA- and DNA-directed DNA polymerase and RNase H activities of both AMV enzymes. Rifampicin was inactive at 100 μg/ml. When used against a variety of non-polymerizing enzymes such as alkaline phosphatase, glutamate-oxaloacetate transaminase, DNase I, and RNase A, these derivatives were inactive at drug concentrations between 100 and 200 μg/ml. Polynucleotide phosphorylase was inhibited slightly by all three derivatives. These results support the idea that rifamycin SV derivatives with appropriate 3-substituted side-chains are specific inhibitors of nucleic acid polymerizing enzymes.     

Synthesis of quinolinate from D-aspartate in the mammalian liver-Escherichia coli quinolinate synthetase system.

Two proteins (A and B) from $\text{Escherichia coli}$ are required for the synthesis of the NAD precursor quinolinate from aspartate and dihydroxyacetone phosphate. Mammalian liver contains a FAD linked protein which replaces $\text{E. coli}$ B protein for quinolinate synthesis. D-aspartic acid but not L-aspartic acid is a substrate for quinolinic acid synthesis in a system composed of the B protein replacing activity of mammalian liver and $\text{E. coli}$ A protein. In contrast the $\text{E. coli}$ B protein-$\text{E. coli}$ A protein quinolinate synthetase system requires L-aspartic acid as substrate. The previous report that L-aspartate was a substrate in the liver-$\text{E. coli}$ system was due to contamination of commercially available [¹⁴C]L-aspartate with [¹⁴C]D-aspartate. These and other observations suggest that liver B protein is D-aspartate oxidase and $\text{E. coli}$ B protein is L-aspartate oxidase.     

Regulation of the 4-hydroxyphenylacetic acid meta-cleavage pathway in an Acinetobacter sp

Lactate-grown cultures of $\text{Acinetobacter}$ sp. strain 3B-1 synthesize constitutively all enzymes except the 4-hydroxyphenylacetic acid-3-hydroxylase. All enzymes are further synthesized when strain 3B-1 is grown with 4-hydroxyphenylacetic acid. Induction studies with two mutant strains, one defective in the 3-hydroxylase, and the other defective in the dehydrogenase, indicate that 4-hydroxyphenylacetic acid induces the 3-hydroxylase only, and the second metabolite 3,4-dihydroxyphenylacetic acid appears to induce 3,4-dihydroxyphenylacetic acid-2,3-dioxygenase and subsequent enzymes. Thus, the enzymes of the 4-hydroxyphenylacetic acid $\text{meta}$-cleavage pathway are synthesized following at least two sequential inductive events.     

Responsibility of 16S RNA for the stimulation of polypeptide synthesis by spermidine.

From the studies on the spermidine stimulation of polyphenylalanine synthesis catalyzed by $\text{E. coli}$ 50S and reconstituted 30S particles containing 16S RNA and 30S ribosomal proteins from $\text{E. coli}$ and $\text{B. thuringiensis}$ in different kinds of combinations, it is concluded that 16S RNA is mainly responsible for the stimulation of polypeptide synthesis by spermidine.     

Structural alteration of rRNA in the L7/L12 region of 50s ribosome on removal of L7/L12 proteins

Core particles of 50S ribosomes depleted of $\text{L7}\text{L12}$ proteins are degraded by RNase I at a considerably slower rate than intact 50S ribosomes. The normal rate is restored on incorporating $\text{L7}\text{L12}$ proteins into the core particles. The capacity of the core particles to inhibit the RNase I-catalyzed hydrolysis of poly A and to bind ethidium bromide is also greater with core particles than with intact 50S ribosomes. It appears from these results that the region(s) of rRNA in the vicinity of $\text{L7}\text{L12}$ proteins has less ordered structure which, on removal of $\text{L7}\text{L12}$ proteins, becomes more organized. Apparently, binding of $\text{L7}\text{L12}$ proteins to the 50S core leads to the destabilization of double-stranded regions of rRNA.     

Genetic analysis of the structure and function of RNase P fromE. coli

A brief review of the genetic studies on ribonuclease P (RNase P) fromEscherichia coli is presented. Temperature-sensitive mutants ofE. coli defective in tRNA processing were isolated by screening cells which were unable to synthesize a suppressor tRNA at restrictive temperature. Structural analysis of accumulated tRNA precursors showed that the isolated mutants were defective in RNase P activity. Analyses of the mutants revealed that the enzyme is essential for the synthesis of all tRNA molecules in cells and that the enzymes consists of two subunits. Analyses of the isolated mutants revealed a possible domain structure of the RNA subunit of the enzyme.Abbreviations E. coli Escherichia coli - RNase P ribonuclease P     

Trypsin inhibitory activity of a polypeptide isolated from red kidney beans, that also enhances lymphocyte stimulation.

A polypeptide isolated from red kidney beans, $\text{Phaseolus}\text{vulgaris}$, which has previously been shown to stimulate RNA synthesis in cultures of mouse spleen lymphocytes and plasmolyzed $\text{E.}\text{coli}$, is here shown to be a potent inhibitor of trypsin and α-chymotrypsin. This polypeptide is compared with commercially available trypsin inhibitors with regard to their capacity to inhibit some proteolytic enzymes and to stimulate $\text{in}\text{vitro}$ cultures of lymphocytes. Similar to FV the lima been trypsin inhibitor was found to possess a stimulating effect on the RNA as well as the DNA synthesis in lymphocyte cultures.     

Translation of AKR-murine leukemia viral RNA in an E. coli cell-free system

High molecular weight RNA isolated from the oncogenic type C murine leukemia virus, AKR-MuLV, stimulates the incorporation of radioactive amino acids into protein in an $\text{E. coli}$ cell-free system. Analysis of the translational products by SDS polyacrylamide gel electrophoresis demonstrated the synthesis of at least three proteins corresponding in molecular weight to several authentic viral proteins. Positive immunoprecipitation tests also confirm the translational product as AKR-MuLV related. Although at least 18 proteins were found on analysis of disrupted murine leukemia virions, only three were synthesized $\text{in vitro}$ in response to AKR-MuLV RNA in the $\text{E. coli}$ cell-free system.     

Increased production of the outer membrane receptors for colicins B, D and M by Escherichia coli under iron starvation.

Growth of $\text{E. coli}$ K-12 under severe iron stress results in increased production of the outer membrane receptors for colicins B, D, Ib and M. The increase in colicin receptor activity coincides with the appearance of large amounts of two high molecular weight proteins in the outer membrane of the cells. These proteins are identified as the outer membrane receptors for colicins B and D and for colicin M. Mutants lacking a functional outer membrane receptor for colicins B and D are defective in the uptake of iron complexed with the siderochrome enterochelin, and are thus comparable with $\text{tonA}$ mutants which lack a functional receptor for colicin M and are defective in the uptake of iron complexed with ferrichrome (6). The colicin B and D receptor may therefore function in the uptake of ferri-enterochelin.     

Bacteriophage T4 tail length is controlled by its baseplate

Purified T4 baseplate, when treated with high concentrations of pancreatic RNase, are inactive in $\text{in}\text{vitro}$ complementation assays. Their ability to initiate tail tube assembly is not altered; but the most probably length of the tube-baseplate formed is only 800A, compared to 1000A, the normal tube length, when untreated baseplates are used. Thus, baseplates help to regulate tube length, possibly by a template mechanism. Several minor baseplate proteins which may be involved in determining the length, including gp54, are missing from RNase - treated base-plates. These effects may be due to an unidentified protease contaminant of the RNase, since they are inhibited by phenylmethane sulfonyl fluoride.     

Regulation of cyclopropane fatty acid biosynthesis by variations in enzyme activities

It is proposed that cyclopropane fatty acid biosynthesis in $\text{Lactobacillus plantarum}$ is regulated by $\text{in vivo}$ variations in the activities of two enzymes acting sequentially. S-adenosylhomocysteine hydrolase relieves the end-product inhibition of cyclopropane synthetase by degrading a product (S-adenosyl-homocysteine) of the latter enzyme activity. Both enzymes show an abrupt increase and subsequent decrease in activity at a time during the bacterial growth cycle which corresponds to the period of most rapid synthesis of cyclopropane fatty acid $\text{in vivo}$.     

The isolation and identification of juvenile hormone from cockroach corpora allata in vitro

Methyl (2E, 6E) - 10, 11 - epoxy - 3, 7, 11 - trimethyl - 2, 6 - dodecadienoate (JH III) is synthesized by corpora cardiaca/allata of the American cockroach $\text{Periplaneta americana in vitro}$ and released into the culture medium. It constitutes the principal part of the biologically detectable JH activity. A fully synthetic medium without terpenoid precursors or serum was used, which proves, that the entire molecule is synthesized $\text{de novo}$. The presence of JH III in orders as different as Blattodea, Orthoptera, Coleoptera and Lepidoptera indicates, that it is the most widely occuring JH of insects.     

Mutational alteration of Bacillus subtilis DNA polymerase 3 to hydroxyphenylazopyrimidine resistance: polymerase 3 is necessary for DNA replication

A spontaneous mutant of $\text{Bacillus}\text{subtilis}$ resistant to killing by two hydroxyphenylazopyrimidines has been isolated. The DNA polymerase III of this mutant is resistant to inhibition by these drugs. The K_i for 6-(p-hydroxyphenylazo)-uracil (HPUra) is 20 μM, about 40 times higher than the K_i of the wild-type enzyme. The mutant and wild-type polymerases behave similarly during purification, are sensitive to N-ethylmaleimide and to 0.1 M KCl, and have the same K_m for dGTP (0.5 μM). The HPUra inhibition of both enzymes is attenuated competitively by dGTP. We conclude that polymerase III is the target for hydroxyphenylazopyrimidines $\text{in}\text{vivo}$, and since the drugs specifically inhibit replicative DNA synthesis, polymerase III is necessary for DNA replication.