Translational control of the expression of the β subunit gene of E., coli RNA polymerase

Using the plasmid pNF1337 as template, a mRNA preparation has been obtained that directs the $\text{in}\text{,}\text{vitro}$ synthesis of fMet-Val, the N-terminal dipeptide of the β subunit of RNA polymerase. RNA polymerase holoenzyme specifically inhibits the mRNA-directed synthesis of fMet-Val showing that the autoregulation by RNA polymerase of β,β′ synthesis is at the level of translation. L factor ($\text{nusA}$ gene product) stimulates the synthesis of fMet-Val from a DNA template but not from mRNA. Rifampicin has no effect on the mRNA-directed synthesis of fMet-Val or the ability of RNA polymerase to inhibit fMet-Val synthesis.     

DNA-dependent in vitro synthesis of Escherichia coli ribosomal protein L10 and the formation of an L10L12 complex

The $\text{in vitro}$ synthesis of ribosomal protein L10 has been demonstrated using λrif^d18 DNA as template. The L10 synthesized $\text{in vitro}$ forms a complex with ribosomal protein L12 and the L10 in this complex can be immunoprecipitated with L12 antiserum.     

Selection of repeated sequences of homologous and heterologous DNA during in vitro transcription by maize RNA polymerase

RNA synthesized $\text{in}\text{vitro}$ by maize RNA polymerase II arises in part from repeated DNA sequences, since significant hybridization to the parent DNA occurs with low concentrations of RNA and DNA. Over three times as much “repeated sequence” RNA is transcribed from maize as from calf thymus DNA.     

Cell-free synthesis of the enzymes of peroxisomal beta-oxidation

Three enzymes of peroxisomal β-oxidation of rat liver were synthesized in a cell-free protein-synthesizing system derived from rabbit reticulocyte lysate. The $\text{in}\text{vitro}$ products of acyl-CoA oxidase and enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase multifunctional protein were similar in size to or slightly larger than the subunit of the respective mature enzymes. The $\text{in}\text{vitro}$ product of peroxisomal 3-ketoacyl-CoA thiolase was about 3,000 daltons larger than the mature subunit. The hepatic levels of translatable mRNAs coding for these three enzymes were about 10 times higher in rats fed a di(2-ethylhexyl)phthalate-containing diet than in control animals.     

Appearance of a kidney-specific ribosomal protein during mouse embryonic development

Ribosomes were prepared from adult mouse liver and kidney and the protein components examined by SDS polyacrylamide gel electrophoresis. A kidney specific protein was identified and was found to be associated with the large ribosomal subunit. $\text{In}$$\text{vitro}$ labelling of 11- and 14-day embryonic kidneys and subsequent analysis of the ribosomal proteins indicated a change in the ribosomal population during development. The kidney specific protein was synthesized during the first four days of kidney organogenesis.     

RNA-protein interactions in the ribosome: VIII. Co-operative interactions in the 50 S subunit of Escherichia coli

Six 50 S ribosomal subunit proteins, each unable to interact independently with the 23 S RNA, were shown to associate specifically with ribonucleoprotein complexes consisting of intact 23 S RNA, or fragments derived from it, and one or more RNA-binding proteins. In particular, L21 and L22 depend for attachment upon L20 and L24, respectively; L5, L10 and L11 interact individually with complexes containing L2 and L16; and one or both proteins of the $\text{L}\text{17}\text{L}\text{27}$ mixture are stimulated to bind in the presence of L1, L3, L6, L13 and L23. Moreover, L14 alone was found to interact with a fragment from the 3′ end of the 23 S RNA, even though it cannot bind to 23 S RNA. By correlating the data reported here with the findings of others, it has been possible to formulate a partial in vitro assembly map of the Escherichia coli 50 S subunit encompassing both the 5 S and 23 S RNAs as well as 21 of the 34 subunit proteins.     

Effects of 3′-deoxyadenosine triphosphate on in vitro RNA synthesis of plant RNA-dependent RNA polymerases

3′-deoxyadenosine triphosphate inhibited $\text{in}\text{vitro}$ [³H]UMP incorporation by RNA-dependent RNA polymerases from tobacco and cowpea plants. The inhibition of [³H]UMP incorporation could be reversed by simultaneous addition of higher ATP concentrations but not with increasing concentrations of UTP or when excess ATP was added 10 min after the inhibitor. These results suggest 3′-deoxyadenosine triphosphate competes specifically with ATP in reaction mixtures and results in premature termination of RNA synthesis $\text{in}\text{vitro}$ by RNA-dependent RNA polymerase.     

Detection of a DNA polymerase β stimulating protein in hela cell nuclear extracts

HeLa cell nuclei contain a protein which stimules the $\text{in}\text{vitro}$ activity of HeLa cell DNA polymerase β, but does not affect the activity of DNA polymerase α and γ. The protein, which binds to both single- and double-stranded DNA, does not possess nuclease activity and is heat stable, surviving 100 degrees C for 10 min. The molecular weight of the protein is approximately 85,000 and evidence is presented that it may exert its stimulatory effect by direct interaction with β-polymerase.     

Altered restriction of nuclear RNA during incubation in vitro

Nuclei were isolated from rat liver and incubated $\text{in}\text{vitro}$ in two commonly employed RNA transport assays. Released [¹⁴C] RNA was isolated and hybridized with filter-bound DNA in the presence of competing cytoplasmic RNA. A significant portion of RNA which was transported to either medium was not represented in cytoplasmic RNA. These results indicate that the restriction of some sequences to the nucleus $\text{in}\text{vivo}$ is not maintained $\text{in}\text{vitro}$.     

Localization of the structural gene for the ' subunit of RNA polymerase in Escherichia coli

A minicell-producing strain of $\text{E.}$$\text{coli}$ carrying an F′ factor, KLF10-1, forms minicells that contain plasmid but not chromosomal DNA. These minicells were found to synthesize two polypeptides corresponding precisely to the β and β′ subunits of RNA polymerase in SDS-polyacrylamide gel electrophoresis. In contrast, minicells obtained from an isogenic strain carrying F13-1 do not synthesize these proteins under similar conditions. These results indicate that the structural genes for the β′ as well as β subunits of the polymerase are located on the chromosomal segment (78 to 81 min on the standard genetic map of $\text{E.}$$\text{coli}$) carried by KLF10-1.     

RNA-protein interactions in the ribosome. II. Binding of ribosomal proteins to isolated fragments of the 16 S RNA

Specific fragments of the 16 S ribosomal RNA of Escherichia coli have been isolated and tested for their ability to interact with proteins of the 30 S ribosomal subunit. The 12 S RNA, a 900-nucleotide fragment derived from the 5′-terminal portion of the 16 S RNA, was shown to form specific complexes with proteins S4, S8, S15, and S20. The stoichiometry of binding at saturation was determined in each case. Interaction between the 12 S RNA and protein fraction $\text{S}\text{16}\text{S}\text{17}$ was detected in the presence of S4, S8, S15 and S20; only these proteins were able to bind to this fragment, even when all 21 proteins of the 30 S subunit were added to the reaction mixture. Protein S4 also interacted specifically with the 9 S RNA, a fragment of 500 nucleotides that corresponds to the 5′-terminal third of the 16 S RNA, and protein S15 bound independently to the 4 S RNA, a fragment containing 140 nucleotides situated toward the middle of the RNA molecule. None of the proteins interacted with the 600-nucleotide 8 S fragment that arose from the 3′-end of the 16 S RNA.When the 16 S RNA was incubated with an unfractionated mixture of 30 S subunit proteins at 0 °C, 10 to 12 of the proteins interacted with the ribosomal RNA to form the reconstitution intermediate (RI) particle. Limited hydrolysis of this particle with T₁ ribonuclease yielded 14 S and 8 S subparticles whose RNA components were indistinguishable from the 12 S and 8 S RNAs isolated from digests of free 16 S RNA. The 14 S subparticle contained proteins S6 and S18 in addition to the RNA-binding proteins S4, S8, S15, S20 and $\text{S}\text{16}\text{S}\text{17}$. The 8 S subparticle contained proteins S7, S9, S13 and S19. These findings serve to localize the sites at which proteins incapable of independent interaction with 16 S RNA are fixed during the early stages of 30 S subunit assembly.     

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.