Studies on the synthesis of cytochrome P-450 and cytochrome P-448 in rat liver

The synthesis of cytochrome P-450 (phenobarbital inducible) and cytochrome P-448 (3-methylcholanthrene inducible) have been studied in rat liver in vivo and in the wheat germ cell-free system using anti- cytochrome P-450 and anti-cytochrome P-448 antibodies. The major mature forms synthesized in vivo correspond to a molecular weight of 47,000 for cytochrome P-450 and 53,000 for cytochrome P-448. Translation of poly(A)-containing RNA from phenobarbital-treated rats in the wheat germ cell-free system reveals that the cell-free product immunoprecipitated with anti-cytochrome P-450 antibody has a molecular weight close to 47,000. In the case of 3-methylcholanthrene, the cell- free product immunoprecipitated with anti-cytochrome P-448 antibody shows a molecular weight around 59,000. Significant conversion of the 59,000 species to the 53,000 species can be demonstrated when the translation is carried out in the presence of microsomal membranes isolated from rat liver. Phenobarbital and 3-methylcholanthrene enhance the translatable messenger.

Number and proportion of selenocucleosides in the transfer RNA of escherichia-coli

tRNA isolated from escherichia-coli grown in a medium containing [75Se] sodium selenosulfate was converted to nucleosides and analysed for selenonucleosides on a phosphocellulose column. Upon chromatography of the nucleosides on phosphocellulose column, the radioactivity resolved into three peaks. The first peak consisted of free selenium and traces of undigested nucleotides. The second peak was identified as 4-selenouridine by co-chromatographing with an authentic sample of 4-selenouridine. The identity of the third peak was not established. The second and third peaks represented 93% and 7% of the selenium present in nucleosides respectively.

Wave analysis of sound decay in rectangular rooms

The decay of sound in a rectangular room is analyzed for various boundary conditions on one of its walls. It is shown that the decay of the sound-intensity level is in general nonlinear. But for specific areas and impedances of the material it is possible to obtain a linear initial decay. It is also shown that the coefficients derived from the initial decay rates neither correspond to the predictions of Sabine's or Eyring's geometrical theories nor to the normal coefficients of Morse's wave theory. The dependence of the coefficients on the area of the material is discussed. The influence of the real and the imaginary parts of the specific acoustic impedance of the material on the coefficients is also discussed. Finally, the existence of a linear initial decay corresponding to the decay of a diffuse field in the case of a highly absorbing material partially covering a wall is explained on the basis of modal coupling.

Number and proportion of selenocucleosides in the transfer RNA of Image Image

tRNA isolated from . grown in a medium containing [75Se] sodium selenosulfate was converted to nucleosides and analysed for selenonucleosides on a phosphocellulose column. Upon chromatography of the nucleosides on phosphocellulose column, the radioactivity resolved into three peaks. The first peak consisted of free selenium and traces of undigested nucleotides. The second peak was identified as 4-selenouridine by co-chromatographing with an authentic sample of 4-selenouridine. The identity of the third peak was not established. The second and third peaks represented 93% and 7% of the selenium present in nucleosides respectively.

Wave analysis of sound decay in rectangular rooms

The decay of sound in a rectangular room is analyzed for various boundary conditions on one of its walls. It is shown that the decay of the sound-intensity level is in general nonlinear. But for specific areas and impedances of the material it is possible to obtain a linear initial decay. It is also shown that the coefficients derived from the initial decay rates neither correspond to the predictions of Sabine's or Eyring's geometrical theories nor to the normal coefficients of Morse's wave theory. The dependence of the coefficients on the area of the material is discussed. The influence of the real and the imaginary parts of the specific acoustic impedance of the material on the coefficients is also discussed. Finally, the existence of a linear initial decay corresponding to the decay of a diffuse field in the case of a highly absorbing material partially covering a wall is explained on the basis of modal coupling.

RNA polymerase activity in isolated nuclei of Nicotiana sanderae callus: Characteristics and modulation during differentiation

Isolated nuclei from differentiating cultures of Nicotiana sanderae showed increased levels of RNA polymerase activity as compared to the nuclei from callus cultures. The RNA synthetic activity was dependent on nucleotide triphosphates and Mg2+ and was destroyed by RNase. Maximum activity was obtained in the presence of 50 mM (NH4)2 SO4 and α-amanitin inhibited 40% and 55% of the activity in the nuclei from callus and differentiating tissue respectively. The nuclei from differentiating tissue elicited a 3-fold increase in RNA polymerase I and a 4-fold augmentation in RNA polymerase II activities.

Two forms of methionyl-transfer RNA synthetase from Mycobacterium Smegmatis

Two methionyl-transfer RNA synthetases (A and B forms) have been isolated from Image . The homogeneous preparations of the enzymes showed 1500 fold increase in specific activity in aminoacylation of methionine specific tRNA. The A and B forms differed in their specificity of aminoacylation of tRNAmMet and tRNAfMet; enzyme B exhibited much higher specificity for tRNAfMet. The molecular activities of A and B enzymes for aminoacid and tRNA were identical. The turnover number for aminoacid was 27 fold greater than that for tRNA, while the Km values for tRNA were lower by a factor of 106 as compared to the aminoacid. Both the enzymes catalysed ATP-pyrophosphate exchange reaction to the same extent.

Nucleotide sequence of the 3' terminal region of belladonna mottle virus-Iowa (renamed Physalis mottle virus) RNA and an analysis of the relationships of tymoviral coat proteins

The 3prime terminal 1255nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3prime terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.

Sequence Specific Interaction between Promoter DNA and Escherichia coli RNA Polymerase: Comparative Thermodynamic Analysis with One Immobilized Partner

Sequence specific interaction between DNA and protein molecules has been a subject of active investigation for decades now. Here, we have chosen single promoter containing bacteriophage Delta D-III T7 DNA and Escherichia coli RNA polymerase and followed their recognition at the air-water interface by using the surface plasmon resonance (SPR) technique, where the movement of one of the reacting species is restricted by way of arraying them on an immobilized support. For the Langmuir monolayer studies, we used a RNA polymerase with a histidine tag attached to one of its subunits, thus making it an xcellent substrate for Ni(II) ions, while the SPR Studies were done using biotin-labeled DNA immobilized on a streptavidin-coated chip. Detailed analysis of the thermodynamic parameters as a function of concentration and temperature revealed that the interaction of RNA polymerase with T7 DNA is largely entropy driven (83 (+/- 12) kcal mol(-1)) with a positive enthalpy of 13.6 (+/- 3.6) kcal mol(-1), The free energy of reaction determined by SPR and Langmuir-Blodgett technique was -11 (+/- 2) and -15.6 kcal mol(-1), respectively. The ability of these methods to retain the specificity of the recognition process was also established.

Synthesis of leader RNA and editing of P mRNA during transcription by rinderpest virus

Purified rinderpest virus was earlier shown to transcribe in vitro, all virus-specific mRNAs with the promoter-proximal N mRNA being the most abundant. Presently, this transcription system has been shown to synthesize full length monocistronic mRNAs comparable to those made in infected cells. Small quantities of bi- and tricistronic mRNAs are also synthesized. Rinderpest virus synthesizes in vitro, a leader RNA of not, vert, similar 55 nucleotides in length. Purified rinderpest virus also exhibits RNA editing activity during the synthesis of P mRNA as shown by primer extension analysis of the mRNA products.

Purification of RNA polymerase from mycobacteria for optimized promoter-polymerase interactions

In vitro transcription analysis is important to understand the mechanism of transcription. Various assays for the analysis of initiation, elongation and termination form the basis for better understanding of the process. Purified RNA polymerase (RNAP) with high specific activity is necessary to carry out variety of these specific reactions. The RNAP purified from Mycobacterium smegmatis from exponential phase showed low promoter specificity in promoter-polymerase interaction studies. This is due to the presence of a large number of sigma factors during exponential phase and under-representation of sigma(A) required for house-keeping transcription. We describe an in vivo reconstitution of RNAP holoenzyme with sigma(A) and its purification, which resulted in holoenzyme with stoichiometric sigma(A) content. The reconstituted holoenzyme showed enhanced promoter-specific binding and promoter-specific-transcription activity compared to the enzyme isolated using standard procedure. Such in vivo reconstitution of stoichiometric holoenzyme could facilitate promoter-specific transcription assays, especially in organisms which encode a large number of sigma factors.

Differential Mechanisms of Binding of Anti-Sigma Factors Escherichia coli Rsd and Bacteriophage T4 AsiA to E. coli RNA Polymerase Lead to Diverse Physiological Consequences

Anti-sigma factors Escherichia coli Rsd and bacteriophage T4 AsiA bind to the essential housekeeping sigma factor, sigma(70), of E. coli. Though both factors are known to interact with the C-terminal region of sigma(70), the physiological consequences of these interactions are very different. This study was undertaken for the purpose of deciphering the mechanisms by which E. coli Rsd and bacteriophage T4 AsiA inhibit or modulate the activity of E. coli RNA polymerase, which leads to the inhibition of E. coli cell growth to different amounts. It was found that AsiA is the more potent inhibitor of in vivo transcription and thus causes higher inhibition of E. coli cell growth. Measurements of affinity constants by surface plasmon resonance experiments showed that Rsd and AsiA bind to or 70 with similar affinity. Data obtained from in vivo and in vitro binding experiments clearly demonstrated that the major difference between AsiA and Rsd is the ability of AsiA to form a stable ternary complex with RNA polymerase. The binding patterns of AsiA and Rsd with sigma(70) studied by using the yeast two-hybrid system revealed that region 4 of sigma(70) is involved in binding to both of these anti-sigma factors; however, Rsd interacts with other regions of sigma(70) as well. Taken together, these results suggest that the higher inhibition of E. coli growth by AsiA expression is probably due to the ability of the AsiA protein to trap the holoenzyme RNA polymerase rather than its higher binding affinity to sigma(70).

RNA triphosphatase and guanylyl transferase activities are associated with the RNA polymerase protein L of rinderpest virus

Rinderpest virus (RPV) large (L) protein is an integral part of the ribonucleoprotein (RNP) complex of the virus that is responsible for transcription and replication of the genome. Previously, we have shown that recombinant L protein coexpressed along with P protein (as the L-P complex) catalyses the synthesis of all viral mRNAs in vitro and the abundance of mRNAs follows a gradient of polarity, similar to the occurrence in vivo. In the present work, we demonstrate that the viral mRNAs synthesized in vitro by the recombinant L or purified RNP are capped and methylated at the N-7 guanine position. RNP from the purified virions, as well as recombinant L protein, shows RNA triphosphatase (RTPase) and guanylyl transferase (GT) activities. L protein present in the RNP complex catalyses the removal of gamma-phosphate from triphosphate-ended 25 nt RNA generated in vitro representing the viral N-terminal mRNA 5' sequence. The L protein forms a covalent enzyme-guanylate intermediate with the GMP moiety of GTP, whose formation is inhibited by the addition of pyrophosphate; thus, it exhibits characteristics of cellular GTs. The covalent bond between the enzyme and nucleotide is acid labile and alkali stable, indicating the presence of phosphoamide linkage. The C-terminal region (aa 1717-2183) of RPV L protein alone exhibits the first step of GT activity needed to form a covalent complex with GMP, though it lacks the ability to transfer GMP to substrate RNA. Here, we describe the biochemical characterization of the newly found RTPase/GT activity of L protein.

Identification and characterization of DdRPB4, a subunit of Dictyostelium discoideum RNA polymerase II

Rpb4, the fourth largest subunit of the eukaryotic RNA polymerase II (RNAPII), is required for growth at extreme temperatures and for an appropriate response to nutrient starvation in yeast. Sequence homologs of Rpb4 are found in most sequenced genomes from yeast to humans. To elucidate the role of this subunit in nutrient starvation, we chose Dictyostelium discoideum, a soil amoeba, which responds to nutrient deprivation by undergoing a complex developmental program. Here we report the identification of homolog of Saccharomyces cerevisiae RPB4 in D. discoideum. Localization and complementation studies suggest that Rpb4 is functionally conserved. DdRPB4 transcript and protein levels are developmentally regulated. Although DdRPB4 could not be deleted, overexpression revealed that the Rpb4 protein is essential for cell survival and is regulated stringently at the post-transcriptional level in D. discoideum. Thus maintaining a critical level of Rpb4 is important for this organism.

Role of 16S ribosomal RNA methylations in translation initiation in Escherichia coli

Translation initiation from the ribosomal P-site is the specialty of the initiator tRNAs (tRNA(fMet)). Presence of the three consecutive G-C base pairs (G29-C41, G30-C40 and G31-C39) in their anticodon stems, a highly conserved feature of the initiator tRNAs across the three kingdoms of life, has been implicated in their preferential binding to the P-site. How this feature is exploited by ribosomes has remained unclear. Using a genetic screen, we have isolated an Escherichia coli strain, carrying a G122D mutation in folD, which allows initiation with the tRNA(fMet) containing mutations in one, two or all the three G-C base pairs. The strain shows a severe deficiency of methionine and S-adenosylmethionine, and lacks nucleoside methylations in rRNA. Targeted mutations in the methyltransferase genes have revealed a connection between the rRNA modifications and the fundamental process of the initiator tRNA selection by the ribosome.