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U Maitra

Publications and source records attributed to U Maitra.

At least 55 records · Page 3Linked to original sources
PubMed1984

Ribosomal subunit antiassociation activity in rabbit reticulocyte lysates. Evidence for a low molecular weight ribosomal subunit antiassociation protein factor (Mr = 25,000).

A ribosomal subunit antiassociation activity has been purified from both the postribosomal supernatant and ribosomal salt-wash protein fractions of rabbit reticulocyte lysates. A majority (greater than 90%) of the activity is associated with a low molecular weight protein of Mr of approximately 25,000. A small but significant level of antiassociation activity (less than 10%) was found to be associated with higher molecular weight protein fractions. The purified 25,000-dalton antiassociation factor interacts with 60 S ribosomal subunits to prevent them from reassociating with 40 S ribosomal subunits. The factor does not seem to interact directly with 40 S subunits nor does it dissociate 80 S monosomes. The properties of this factor are thus similar to the eukaryotic initiation factor 6 isolated from both wheat germ and calf liver extracts.

Animals↗
PubMed1984

Locations and nucleotide sequences of three major class III promoters for bacteriophage T3 RNA polymerase on T3 DNA.

The DNA sequences of three major class III T3 RNA polymerase promoters located at 45.0, 55.0, and 64.8% on the standard T3 genetic map have been determined. The precise RNA initiation sites were also determined by 5'-terminal RNA sequence analysis of the transcripts synthesized from the promoter-containing DNA fragments. Alignment of these three class III promoters and a previously determined T3 RNA polymerase promoter at 1.05% on T3 genetic map, with start points of transcription (+1) in register, indicates a high degree of sequence conservation among the four T3 RNA polymerase promoters. The sequences are identical between positions -12 and +4 and are uniformly A-T between -12 and -17. The conserved portion of the (-) strand sequence is 5' (sequence in text) Upstream from -17 and downstream from +4 the sequences diverge. Comparison of this sequence with a prototype 23-base pair promoter sequence for T7 RNA polymerase shows overall homology between positions -17 and +4 with conserved divergence at residues -2 and between -10 and -12. Furthermore, careful examination of the nucleotide sequences around 45.0 and 64.8 T3 map units shows that the putative RNA sequences arising from these regions by overlapping transcription from upstream promoters can be arranged into stable stemloop structures thought to be required for RNase III cleavage. This pattern is similar to those reported for the corresponding T7 RNA polymerase promoters on T7 DNA (Dunn. J. J., and Studier, F. W. (1983) J. Mol. Biol. 166, 477-535).

Base Sequence↗
PubMed1982

Eukaryotic ribosomal subunit anti-association activity of calf liver is contained in a single polypeptide chain protein of Mr = 25,500 (eukaryotic initiation factor 6).

A protein factor that prevents the reassociation of eukaryotic 40 S and 60 S ribosomal subunits when the Mg+ concentration is raised from 1 to 5 mM has been purified to apparent electrophoretic homogeneity from postribosomal supernatant of calf liver extracts. The purified ribosomal subunit anti-association factor is a relatively heat-sensitive protein consisting of a single polypeptide chain of apparent Mr = 25,500. Direct assay for ribosomal subunit anti-association activity indicates that the majority (greater than 90% of such an activity in calf liver extracts can be accounted for by the presence of this 25,500-dalton protein factor in the postribosomal supernatant. The ribosomal salt wash protein fractions are virtually devoid of any significant ribosomal subunit anti-association or 80 S ribosome dissociation activity. The purified anti-association factor maintains a pool of ribosomal subunits by binding to 60 S ribosomal subunits and preventing them from reassociating with 40 S ribosomal subunits, rather than by dissociating 80 S monosomes. The factor neither binds to, nor seems to interact directly with, 40 S subunits. The properties of this factor are thus similar to wheat germ ribosome dissociation factor (eukaryotic initiation factor 6) described by Russell and Spremulli ((1979) J. Biol. Chem. 254, 8796-8800.

Animals↗
PubMed1981

Location, function, and nucleotide sequence of a promoter for bacteriophage T3 RNA polymerase.

The major promoters for bacteriophage T3 RNA polymerase on the T3 genome have been mapped by DNA.RNA filter hybridization. One promoter is located in a 300-base-pair Hpa I restriction fragment near the genetic "left" end of T3 DNA. The sequence in the vicinity of the major initiation site of transcription in this region has been determined. A part of the (-)strand sequence is 5' T-A-T-T-T-A-C-C-C-T-C-A-C-T-A-A-A-G-+1 G-G-A-A-U 3'. Comparison of this sequence with the prototype 23-base-pair promoter sequence for bacteriophage T7 RNA polymerase shows a striking pattern of homology and divergence. Between positions -9 and +4, the sequences are virtually identical, whereas between positions -17 and -10, the sequences are quite different. It is postulated that these sequence subsets may perform different functions in transcription initiation by the phage RNA polymerases.

Base Sequence↗
PubMed1980

Construction of a restriction map of bacteriophage T3 DNA.

A restriction endonuclease cleavage map of bacteriophage T3 DNA has been constructed. The enzymes used and, within parentheses, the number of their cleavage sites on T3 DNA are: HindIII (1), XbaI (1), BglII (1), KpnI (2), MboI (9), and HpaI (17). The size and the relative location of each fragment have been established, defining an accurate physical map of T3 DNA.

Chromosome Mapping↗
PubMed1980

Rabbit reticulocyte initiation factor 2 contains two polypeptide chains of molecular weights 48,000 and 38,000.

Eukaryotic initiation factor 2 (eIF-20) purified from rabbit reticulocyte lysates consists of equimolar amounts of two polypeptide chains of Mr 48,000 and 38,000. Determination of the molecular weight of the native factor gave a value which is consistent with a Mr of 86,000 indicating that the factor is composed of one Mr 48,000 and one Mr 38,000 polypeptide. The purified factor exhibited all the binding activities characteristic of eIF-2. The factor formed ternary complexes with Met-tRNAfMet and GTP; it bound GDP to form a binary complex; and it also possessed the property of binding a wide variety of RNA species, including reoviral mRNA, phage T3 mRNA, rRNAs, and tRNA. Furthermore, the ternary complex formed by purified eIF-2 interacted with the 40S ribosomal subunit in the presence of AUG codon to form a 40S initiation complex. These results indicate that all binding activities attributed to eIF-2 are contained in the 48,000- and 38,000-dalton polypeptides.

Animals↗
PubMed1980

Isolation and sequence determination of 5'-terminal oligonucleotide fragments of RNA transcripts synthesized by bacteriophage T3-induced RNA polymerase from T3 DNA.

The nucleotide sequence of the 5'-terminal oligonucleotides produced by pancreatic RNase digestion of bacteriophage T3 RNA polymerase (EC 2.7.7.6) transcripts of T3 DNA has been determined. The sequence determination is based upon a simple isolation procedure for the 5'-terminal oligonucleotides. This procedure involves treatment of pancreatic RNase digests of alpha 32P-labeled T3 RNA polymerase transcripts with bovine brain exoribonuclease to remove oligonucleotides with free 5'-hydroxyl termini and then chromatographing the products on hydroxylapatite to resolve the remaining oligonucleotides having 5'-phosphate termini. By application of standard two-dimensional separation and sequence techniques, the major 5'-end sequences deduced were pppGpGpGpApGpApGpApY(Y = pyrimidine nucleoside) and pppGpGpGpApGpApCp. In addition, the sequences of other minor 5'-terminal oligonucleotides observed on homochromatograms were also determined. The sequences of these 5'-oligonucleotides were pppGpGpGpApApCpY, pppGpGpGpApApUpY, pppGpGp(2-4 Gp, 2-3 ApGp)..., and pppGpGpGp.... These results demonstrate that T3 phage-induced RNA polymerase possesses a high degree of specificity in the initiation of RNA chains.

Base Sequence↗
PubMed1979

Purified eukaryotic initiation factor 2 from calf liver consists of two polypeptide chains of 48,000 and 38,000 daltons.

Eukaryotic initiation factor 2 (eIF-2), which specifically binds Met-tRNAMetf and forms stable ternary complexes with GTP, has been purified from ribosomal salt wash proteins from calf liver. The purified factor exhibits only two polypeptide bands of Mr = 48,000 and 38,000 following electrophoresis in 15% polyacrylamide gels in the presence of sodium dodecyl sulfate. Densitometric tracings show the two polypeptides are present in a molar ratio of 1:1. This suggests a Mr = 86,000 for the native enzyme, a value which agrees with the apparent molecular weight determined by physical methods. Less pure preparations of eIF-2 show additional polypeptide bands, including 50,000- and 46,000-dalton bands, all of which can be removed by further purification without affecting the activity of eIF-2.

Animals↗
PubMed1979

Termination of transcription by bacteriophage T3 RNA polymerase: homogeneous 3'-terminal oligonucleotide sequence of in vitro T3 RNA polymerase transcripts.

RNA was synthesized in vitro from a T3 DNA template by T3 RNA polymerase and subsequently separated into seven discrete size classes (molecular weights ranging between 0.21 x 10(6) and 6.2 x 10(6)) by electrophoresis in polyacrylamide slab gels. RNase T1-generated 3'-terminal oligonucleotide fragments were then selectively isolated from either the unfractionated total RNA or the gel-purified specific transcripts by chromatography on columns of dihydroxyboryl-cellulose. Sequence analysis of these oligonucleotide products indicated that the unfractionated transcripts as well as all the individual major RNA species examined had a unique sequence, (Gp)UpUpUpUpUpGOH, at their 3' termini. The specificity of this sequence, as well as the total lack of any sequence heterogeneity at the ends of these transcripts, indicates a high degree of specificity of termination during transcription in this system.

Base Sequence↗
PubMed1979

Termination of transcription by Escherichia coli RNA polymerase: influence of secondary structure of RNA transcripts on rho-independent and rho-dependent termination.

The effect of RNA secondary structure on rho-independent and rho-dependent termination of transcription of T3 DNA by Escherichia coli RNA polymerase has been studied by incorporating, into nascent transcripts, base analogs that lead to altered base-pairing properties. A guanine --> hypoxanthine substitution, with attendant weakening of secondary structure, abolished the rho-independent termination at 20% of the genome; in contrast, replacement of cytosine with 5-bromocytosine, which forms stronger pairs with guanine, enhanced termination at this site. rho-Independent termination was not altered by replacing uracil with 5-bromouracil. There are two major rho-dependent termination sites on the T3 DNA-at 8 and 15%. The termination activity of rho in this system also depended on RNA secondary structure. The incorporation of 5-bromouracil instead of uracil into RNA did not alter the site specificity of rho action but rho was rendered inactive when cytosine was replaced by 5-bromocytosine. In contrast, replacement of GTP with ITP in the reaction increased rho-dependent inhibition of RNA synthesis, caused production of heterogeneous-sized transcripts, and stimulated rho-mediated ATP hydrolysis. The rho-associated ATPase activity, in the presence of isolated T3 RNA, was also stimulated by inosine substitution. Furthermore, the temperature-sensitive rho isolated from rho 15 mutant of E. coli, which does not terminate transcription in the presence of the common rNTPs, was active when GTP was replaced with ITP. These results suggest that strongly paired G.C-rich regions in RNA stem-loop structures or RNA.DNA hybrids are essential for rho-independent termination, whereas rho-dependent termination requires weakly paired cytosine residues for its action.

Adenosine Triphosphatases↗
PubMed1977

Function of initiation factor 1 in the binding and release of initiation factor 2 from ribosomal initiation complexes in Escherichia coli.

1. Studies on the function of initiation factor 1 (IF-1) in the formation of 30 S initiation complexes have been carried out. IF-1 appears to prevent the dissociation of initiation factor 2 (IF-2) from the 30 S initiation complex. The factor has no effect on either the initial binding of IF-2 nor does it increase the amount of IF-2 dependent fMet-tRNA and GTP bound to the 30 S subunit. Bound fMet-tRNA remains stable to sucrose gradient centrifugation even in the absence of IF-1. 2. It is postulated that the presence of IF-2 on the 30 S complex is necessary so that at the time of junction with the 50 S subunit to form a 70 S complex, the 70 S-dependent GTPase activity of IF-2 can hydrolyze GTP. This hydrolysis provides a means by which GTP can be removed to facilitate formation of a 70 S initiation complex active in peptidyl transfer. In support of this postulate, it was observed that 30 S initiation complexes formed in the absence of IF-1 could be depleted of their complexes were still able to accept 50 S subunits to form 70 S complexes which could still donate fMet-tRNA into peptide linkages. These results indicate that 30 S complexes lacking GTP do not require IF-2 for formation of active 70 S complexes. 3. IF-1, which is required to prevent dissociation of IF-2 from the 30 S initiation complex, is also required for release of IF-2 from ribosomes following 70 S initiation complex formation. The mechanisms of the release of IF-2 has been studied in greater detail. Evidence is presented which rules out the presence of a stable IF-2 GDP complex on the surface of the 70 S ribosome following GTP hydrolysis and of any exchange reactions between IF-1 and guanine nucleotides necessary for effecting the release of IF-2. IF-2 remains on the 70 S initiation complexes after release of guanine nucleotides and can be liberated solely by addition of IF-1.

Binding Sites↗
PubMed1977

Ribonuclease III cleavage of bacteriophage T3RNA polymerase transcripts to late T3 mRNAs.

In vitro transcription of T3 DNA by T3 phage-induced RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) yields eight discrete RNAs (designated I-VIII) with molecular weights of approximately 6.2, 4.7, 4, 2.8, 1.8, 0.9, 0.52, and 0.21 X 10(6), respectively. Comparison of the size of in vitro T3 RNA polymerase transcripts with in vivo late T3 mRNAs indicates that several late RNAs produced in T3-infected cells do not correspond to any of the in vitro RNAs, and no RNAs as large as the three largest in vitro RNA species, I, II, and III, are observed. Escherichia coli RNase III cleaves these three high molecular weight T3 RNA polymerase transcripts to discrete RNAs that comigrate in polyacrylamide gel electrophoresis with some of the late T3 RNAs.

Coliphages↗
PubMed1976

Interaction of bacterial initiation factor 2 with initiator tRNA.

Interaction of bacterial polypeptide chain initiation factor 2 (IF-2) with Escherichia coli formylmethionyl-tRNA in the absence of free Mg2+ renders the fMet-tRNA adsorable to nitrocellulose membrane filters. This reaction does not require GTP and is strongly inhibited by low concentration (1 mM) of Mg2+ in the reaction mixture. The structural requirements of the tRNA for binary complex formation have been studied using modified fMet-tRNAfMet molecules and a series of N-blocked and normal aminoacyl-rRNAs. It has been observed that IF-2 will not blind either to free formylmethionine or to a short fMet-oligonucleotide, but will bind to any xRNA structure covalently attached to an N-blocked methionine group. The E. coli initiator and noninitiator methiionine tRNAs, which have many differences in primary structure, were found to bind identically. In addition, fMet-tRNAfMet molecules containing structural modidifications at 20 different sites had the same affinity for IF-2 as unmodified fMet-tRNAfMet. N-blocked eukaryotic initiator tRNAs were also found to bind strongly to the factor. Binary complex formation was readily reversible, f[14C]Met-tRNAfMet being competed out by addition of an equal amount of unlabeled fMet-tRNAfMet to the preformed complex. In contrast, deacylated tRNAfMet was a poor compeitor, a 30-fold excess being required for 50% inhibition of complex formation in the presence of limiting factor. Although tRNAs having an N-blocked methionine were found to have the greatest affinity for IF-2, specificity for the amino acid in binary complex formation was not absolute. Partial binding was observed with N-substituted tyrosine, valine, and phenylalanine tRNAs, and weak or no binding with N-subsituted lysine, alanine, and leucine tRNAs. In all cases, N-blocked derivatives had a higher affinity for IF-2 than the corresponding unsubstituted aminoacyl-tRNAs. These results indicate that IF-2 alone is not capable of distinguishing the nucleotide sequence of tRNAs and selects the initiator tRNA by recognizing the fMet moiety. The overall data suggest that the role of IF-2 in formation of the ribosomal initiation complex is to stablize the interaction of fMet-tRNAfMet with the ribosome at low Mg2+ concentrations by binding to both the ribosomal particle and the fMet group of the tRNA.

Amino Acyl-tRNA Synthetases↗