Biomedical subjects

U Maitra

Publications and source records attributed to U Maitra.

At least 37 records · Page 2Linked to original sources

PubMed1996

Characterization of multiple mRNAs that encode mammalian translation initiation factor 5 (eIF-5).

Eukaryotic translation initiation factor 5 (eIF-5) interacts with the 40 S initiation complex (40S.mRNA.MettRNAf.eIF-2.GTP) to promote the hydrolysis of bound GTP with the concomitant joining of the 60 S ribosomal subunit to the 40 S initiation complex to form a functional 80 S initiation complex. In this paper, the multiple mRNAs that encode mammalian eIF-5 have been characterized. In rat tissues, three major eIF-5 mRNAs of 3.5, 2.8, and 2.2 kilobases in length are detected. All major eIF-5 mRNAs are initiated from a single transcription initiation site, contain identical 5'-untranslated and coding regions, but differ from one another only in the length of their 3'-untranslated regions. The different lengths of the 3'-untranslated region of eIF-5 mRNAs are generated by the use of alternative polyadenylation signals. Additionally, we demonstrate tissue-specific variations in eIF-5 mRNA expression as well as preference for polyadenylation sites. These results should lead to increased understanding of the regulation of eIF-5 gene expression.

Alternative Splicing↗

PubMed1996

The A1 x U72 base pair conserved in eukaryotic initiator tRNAs is important specifically for binding to the eukaryotic translation initiation factor eIF2.

The formation of a specific ternary complex between eukaryotic initiation factor 2 (eIF2), the initiator methionyl-tRNA (Met-tRNA), and GTP is a critical step in translation initiation in the cytoplasmic protein-synthesizing system of eukaryotes. We show that the A1 x U72 base pair conserved at the end of the acceptor stem in eukaryotic and archaebacterial initiator methionine tRNAs plays an important role in this interaction. We changed the A1 x U72 base pair of the human initiator tRNA to G1 x C72 and expressed the wild-type and mutant tRNA genes in the yeast Saccharomyces cerevisiae by using constructs previously developed in our laboratory for expression of the human initiator tRNA gene in yeasts. We show that both the wild-type and mutant human initiator tRNAs are aminoacylated well in vivo. We have isolated the wild-type and mutant human initiator tRNAs in substantially pure form, free of the yeast initiator tRNA, and have analyzed their properties in vitro. The G1 x C72 mutation affects specifically the binding affinity of eIF2 for the initiator tRNA. It has no effect on the subsequent formation of 40S or 80S ribosome initiator Met-tRNA-AUG initiation complexes in vitro or on the puromycin reactivity of the Met-tRNA in the 80S initiation complex.

Base Sequence↗

PubMed1994

Purification and characterization of bacterially expressed mammalian translation initiation factor 5 (eIF-5): demonstration that eIF-5 forms a specific complex with eIF-2.

Eukaryotic translation initiation factor 5, eIF-5, has been purified to apparent electrophoretic homogeneity from overproducing Escherichia coli cells expressing the cDNA of the initiation factor under the control of the T7 promoter-T7 RNA polymerase system. Purified recombinant eIF-5 mimics natural eIF-5 isolated from mammalian cells in size, in specific activity, in its ability to catalyze the hydrolysis of GTP bound to the 40S initiation complex, and in the subsequent joining with 60S ribosomal subunits to form the 80S initiation complex. Further characterization of eIF-5 demonstrates that eIF-5 specifically associates with eIF-2, forming an eIF-2.eIF-5 complex. The protein complex sediments in glycerol gradients with an apparent M(r) of 160,000, suggesting that the two proteins associate in a 1:1 stoichiometry. The association between the two initiation factors is highly specific. Addition of 32P-labeled eIF-5 to a partially purified rabbit reticulocyte initiation factor preparation that contained, in addition to eIF-2 and eIF-5, other initiation factors and many other proteins resulted in the specific binding of labeled eIF-5 only to eIF-2, forming a 160-kDa protein complex. In agreement with these observations, we found that in crude initiation factor preparations derived from rabbit reticulocyte lysates, eIF-5 was present as an eIF-2.eIF-5 complex. The significance of eIF-2.eIF-5 complex formation in the overall mechanism of GTP hydrolysis in protein synthesis initiation is discussed.

Animals↗

PubMed1993

Characterization of mammalian translation initiation factor 5 (eIF-5). Demonstration that eIF-5 is a phosphoprotein and is present in cells as a single molecular form of apparent M(r) 58,000.

Eukaryotic translation initiation factor 5 (eIF-5) promotes the hydrolysis of GTP bound to the 40 S initiation complex (40 S.mRNA.Met-tRNA(f).eIF-2.GTP). Using assays that measure (a) the release of 32Pi from [gamma-32P]GTP bound to the 40 S initiation complex and (b) the eIF-5-dependent formation of an 80 S initiation complex from a preformed 40 S initiation complex containing bound [35S]Met-tRNA(f), we devised a novel and rapid procedure for purifying eIF-5 from rabbit reticulocyte lysates in high yield. Highly purified eIF-5 is a monomeric protein with a M(r) of about 58,000. However, in partially purified preparations, 58-kDa eIF-5 is associated with other cellular protein(s) and sediments in glycerol gradient centrifugation as a protein of M(r) approximately 160,000. Chicken antibodies to native eIF-5 were isolated from egg yolks of laying hens immunized with rabbit reticulocyte eIF-5. 35S-Labeled eIF-5, isolated from rat anterior pituitary GH3 cells using affinity-purified anti-eIF-5 antibodies, also has an apparent M(r) of 58,000. eIF-5 immunoprecipitated from extracts of 32P-labeled GH3 cells was phosphorylated on serine residues. Phosphopeptide mapping revealed two major sites of phosphorylation, which are distinct from rabbit reticulocyte eIF-5 sites phosphorylated in vitro by casein kinase II. These results demonstrate that eIF-5 is a phosphoprotein that is present in cells as a single molecular form of apparent molecular weight 58,000.

Animals↗

PubMed1993

Eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae. Cloning, characterization, and expression of the gene encoding the 45,346-Da protein.

Eukaryotic translation initiation factor 5 (eIF-5) catalyzes hydrolysis of GTP bound to a 40 S ribosomal initiation complex with the subsequent joining of a 60 S ribosomal subunit to form an 80 S initiation complex. The yeast gene that encodes eIF-5, designated TIF5, has been isolated and expressed in Escherichia coli to yield a catalytically active eIF-5 protein. TIF5 is a single-copy gene that maps on yeast chromosome XVI and is essential for cell viability. The gene contains an intron-free open reading frame that encodes a protein of calculated M(r) 45,346 in close agreement with the apparent molecular weight of eIF-5 isolated from yeast cells. Sequence analysis of the gene reveals several interesting features. First, the presence of two in-frame translational start sites located 51 base pairs apart suggests the possibility that two proteins, differing by an amino-terminal extension of 17 amino acids, could be generated from the TIF5 gene via differential translational starts. This would explain the presence, in yeast cell lysates, of two forms of eIF-5 differing in molecular weight by about 2,000. Second, the predicted amino acid sequence of eIF-5 contains sequence motifs characteristic of proteins of the GTPase superfamily.

Amino Acid Sequence↗

PubMed1993

Molecular cloning and expression of cDNA for mammalian translation initiation factor 5.

Eukaryotic translation initiation factor 5 (eIF-5) catalyzes the hydrolysis of GTP bound to the 40S ribosomal initiation complex (40S.AUG.Met-tRNAf-eIF-2.GTP) with the subsequent joining of a 60S ribosomal subunit resulting in the formation of a functional 80S initiation complex. A rat cDNA that encodes eIF-5 has been isolated and expressed in Escherichia coli to yield a catalytically active eIF-5 protein. The 3.55-kb cDNA encodes a protein of 429 amino acids (calculated M(r) 48,926) with properties that are similar to eIF-5 isolated from rabbit reticulocyte lysates. The deduced amino acid sequence of eIF-5 contains sequence motifs characteristic of proteins of the GTPase superfamily.

Amino Acid Sequence↗

PubMed1993

Isolation and immunochemical characterization of eukaryotic translation initiation factor 5 from Saccharomyces cerevisiae.

Eukaryotic translation initiation factor 5 (eIF-5), which catalyzes the hydrolysis of GTP bound to the 40 S ribosomal initiation complex has been purified from yeast cell lysates. The purified factor eluted from gel filtration columns as a protein of apparent M(r) = 45,000-50,000. However, when the purified preparation was analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, two distinct polypeptides of apparent M(r) = 54,000 and 56,000 were observed. Each of the two polypeptides individually was found to contain eIF-5 activity, and they were immunologically related to each other. In less pure preparations of yeast eIF-5, however, a significant proportion of eIF-5 activity eluted from gel filtration columns as a protein of M(r) > 140,000. Immunochemical methods were therefore employed to determine the molecular structure of eIF-5 in crude yeast cell lysates. Antisera against purified yeast eIF-5 were prepared in rabbits and shown to be highly potent in inhibiting eIF-5-mediated 80 S initiation complex formation. When crude eIF-5 preparations, as well as yeast cells that were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate, were analyzed by Western blots probed with affinity-purified anti-eIF-5 antibodies, a major immunoreactive polypeptide (apparent M(r) = 54,000) and a minor band (apparent M(r) = 56,000) were observed. No precursor forms of molecular weight higher than 56,000 were detected in any preparations. These results suggest that yeast eIF-5 is a monomeric protein of apparent M(r) = 50,000-56,000.

Animals↗

PubMed1992

Release and recycling of eukaryotic initiation factor 2 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

The eukaryotic initiation factor (eIF)-5 mediates hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. The eIF-2.GDP formed under these conditions is released from the 40 S ribosomal subunit while initiator Met-tRNA(f) remains bound. The released eIF-2.GDP can participate in an eIF-2B-catalyzed GDP/GTP exchange reaction to reform the Met-tRNA(f).eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were also present in an eIF-5-catalyzed reaction, the eIF-2.GDP produced remained bound to the 60 S ribosomal subunit of the 80 S initiation complex. When such an 80 S initiation complex, containing bound eIF-2.GDP, was incubated with GTP and eIF-2B, GDP was released. However, eIF-2 still remained bound to the ribosomes and was unable to form a Met-tRNA(f)l.eIF-2.GTP ternary complex. In contrast, when 60 S ribosomal subunits were preincubated with either free eIF-2 or with eIF-2.eIF-2B complex and then added to a reaction containing both the 40 S initiation complex and eIF-5, the eIF-2.GDP produced did not bind to the 60 S ribosomal subunits but was released from the ribosomes. Thus, the 80 S initiation complex formed under these conditions did not contain bound eIF-2.GDP. Under similar experimental conditions, preincubation of 60 S ribosomal subunits with purified eIF-2B (free of eIF-2) failed to cause release of eIF-2.GDP from the ribosomal initiation complex. These results suggest that 60 S ribosome-bound eIF-2.GDP does not act as a direct substrate for eIF-2B-mediated release of eIF-2 from ribosomes. Rather, the affinity of 60 S ribosomal subunits for either eIF-2, or the eIF-2 moiety of the eIF-2.eIF-2B complex, prevents association of 60 S ribosomal subunits with eIF-2.GDP formed in the initiation reaction. This ensures release of eIF-2 from ribosomes following hydrolysis of GTP bound to the 40 S initiation complex.

Animals↗

PubMed1991

Function of eukaryotic initiation factor 5 in the formation of an 80 S ribosomal polypeptide chain initiation complex.

Eukaryotic initiation factor 5 (eIF-5), isolated from rabbit reticulocyte lysates, is a monomeric protein of 58-62 kDa. The function of eIF-5 in the formation of an 80 S polypeptide chain initiation complex from a 40 S initiation complex has been investigated. Incubation of the isolated 40 S initiation complex (40 S.AUG.Met.tRNAf.eIF-2 GTP) with eIF-5 resulted in the rapid and quantitative hydrolysis of GTP bound to the 40 S initiation complex. The rate of this reaction was unaffected by the presence of 60 S ribosomal subunits. Analysis of eIF-5-catalyzed reaction products by gel filtration indicated that both eIF-2.GDP binary complex and Pi formed were released from the ribosomal complex whereas Met-tRNAf remained bound to 40 S ribosomes as a Met-tRNAf.40 S.AUG complex. Reactions carried out with biologically active 32P-labeled eIF-5 indicated that this protein was not associated with the 40 S.AUG.Met-tRNAf complex; similar results were obtained by immunological methods using monospecific anti-eIF-5 antibodies. The isolated 40 S.AUG.Met-RNAf complex, free of eIF-2.GDP binary complex and eIF-5, readily interacted with 60 S ribosomal subunits in the absence of exogenously added eIF-5 to form the 80 S initiation complex capable of transferring Met-tRNAf into peptide linkages. These results indicate that the sole function of eIF-5 in the initiation of protein synthesis is to mediate hydrolysis of GTP bound to the 40 S initiation complex in the absence of 60 S ribosomal subunits. This leads to formation of the intermediate 40 S.AUG.Met-tRNAf and dissociation of the eIF-2.GDP binary complex. Subsequent joining of 60 S ribosomal subunits to the intermediate 40 S.AUG.Met-tRNAf complex does not require participation of eIF-5. Thus, the formation of an 80 S ribosomal polypeptide chain initiation complex from a 40 S ribosomal initiation complex can be summarized by the following sequence of partial reactions. (40 S.AUG.Met-tRNAf.eIF-2.GTP) eIF-5----(40 S.AUG.Met-tRNAf) + (eIF-2.GDP) + Pi (1) (40 S.AUG.Met-tRNAf) + 60 S----(80 S.AUG.Met-tRNAf) (2) 80 S initiation complex.

Animals↗

PubMed1989

Relative efficiency of utilization of promoter and termination sites by bacteriophage T3 RNA polymerase.

Bacteriophage T3 RNA polymerase promoters have been classified as class II and class III on the basis of their relative location in T3 DNA as well as on the function of the protein products encoded by the messages transcribed from them. In the present work, the efficiency of utilization of several class II and class III promoters by bacteriophage T3 RNA polymerase was compared with regard to (a) rate of initiation of transcription as determined by [32P]PPi exchange with GTP; (b) complex formation between polymerase and promoters in the presence of GTP; and (c) competition between different promoters for T3 RNA polymerase in a standard transcription assay. The results of these experiments indicated that the class II promoters at 1.05 and 22.8 T3 map units, whose promoter sequences are remarkably similar to the consensus class III promoter sequences, are nearly as strong as typical class III promoters. In contrast, the class II promoter at 14.3 T3 map units, whose promoter sequence differs from the consensus class III promoter sequence by having a C:G base pair instead of a usual A:T base pair at the -1 position, was considerably weaker than the class III promoter. When the C:G base pair at this position was changed to A:T using site-directed mutagenesis, the rate of initiation of RNA synthesis from the mutant promoter was similar to that of a typical class III promoter. In agreement with this observation, it was observed that changing the A:T base pair at the -1 position of a strong class II promoter, at 1.05 T3 map units, to C:G decreased the rate of RNA synthesis from this promoter by about 65%. These observations indicate that the nucleotide residues at the -1 position play a critical role in determining the efficiency of promoter utilization by T3 RNA polymerase. The two termination sites recognized in vitro by bacteriophage T3 RNA polymerase on the T3 genome have been cloned, sequenced, and mapped. Analysis of the DNA nucleotide sequence surrounding the termination site at 59.7 map units indicated that the putative RNA transcript arising from this region can be arranged into a GC-rich stem-loop structure followed by a U-rich 3' tail. However, a major fraction of T3 RNA polymerase molecules read through this terminator in vitro to transcribe regions of T3 DNA beyond this terminator. In contrast to termination at 59.7 map units, termination of transcription at 100 T3 map units does not occur in response to any putative terminator structure or sequence.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1989

Further characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Immunochemical characterization and phosphorylation by casein kinase II.

Eukaryotic initiation factor (eIF)-5, isolated from rabbit reticulocyte lysates, is a monomeric protein of Mr = 58,000-62,000. Immunochemical methods were employed to identify eIF-5 in crude cell lysates. Antisera against purified denatured eIF-5 were prepared in rabbits and characterized by immunoblotting and immunoprecipitation techniques using native and denatured eIF-5 as antigens. Monospecific antibodies to denatured eIF-5 were affinity-purified using eIF-5 blotted onto aminophenylthioether paper. Rabbit reticulocytes, HeLa cells and mouse L cells were lysed directly into a denaturing buffer containing 3% sodium dodecyl sulfate. The denatured proteins were analyzed by polyacrylamide gel electrophoresis followed by immunoblotting with anti-eIF-5 antibodies. With each lysate, one major immunoreactive polypeptide was observed whose molecular weight corresponded to that of purified eIF-5 (Mr = 58,000-62,000). No degradation products or precursor forms of molecular weight higher than 62,000 were detected in any lysate. These results indicate that isolated eIF-5 is the same size as that found in crude lysates. Additional characterization of eIF-5 indicates that purified eIF-5 can be phosphorylated at serine residues in vitro by casein kinase II. Furthermore, in vitro phosphorylated eIF-5 retains full biological activity in catalyzing the joining of 60 S ribosomal subunits to a preformed 40 S ribosomal initiation complex to form an 80 S initiation complex. Based on its specific activity, we demonstrate that 1 pmol of rabbit reticulocyte eIF-5 mediates the formation of approximately 180 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.

Animals↗

PubMed1987

Characterization of eukaryotic initiation factor 5 from rabbit reticulocytes. Evidence that the initiation factor is a monomeric protein of Mr of about 58,000-62,000.

Eukaryotic initiation factor 5 (eIF-5) has been purified from the ribosomal salt-wash proteins of rabbit reticulocyte lysates. The purified factor migrates as a single polypeptide upon sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of about 58,000-62,000. In contrast, less pure preparations of reticulocyte eIF-5 behave in gel filtration columns and in glycerol gradient centrifugation in buffers containing 75-100 mM KCl as a protein of apparent Mr = 140,000-160,000. Presumably, this is due to association of the factor with other proteins, since eIF-5 activity present in such preparations can also be shown by (a) glycerol gradient centrifugation in buffers containing 500 mM KCl or (b) gel electrophoresis under denaturing conditions, to be associated with a 58,000-62,000-dalton protein. Furthermore, eIF-5 purified from rabbit reticulocyte lysates in the absence or presence of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity. It can be calculated that 1 pmol of the purified eIF-5 catalyzes the formation of nearly 50 pmol of 80 S initiation complex under in vitro initiation reaction conditions. Because of the highly catalytic activity of eIF-5 in initiation reactions, the presence of even low levels of eIF-5 in eIF-2 preparations causes hydrolysis of GTP bound to the 40 S initiation complex. This results in destabilization of Met-tRNA(f) bound to the 40 S complex in sucrose gradient centrifugation.

Animals↗

PubMed1986

Specific binding of monomeric bacteriophage T3 and T7 RNA polymerases to their respective cognate promoters requires the initiating ribonucleoside triphosphate (GTP).

Bacteriophage T3 and T7 RNA polymerases are monomeric proteins of Mr of about 100,000. Each polymerase has stringent specificity for its own promoters that is present only on the homologous phage DNA template. Neither enzyme recognizes the heterologous phage promoters or Escherichia coli RNA polymerase promoters. In the present study, the interaction of T3 and T7 RNA polymerases with their respective cognate promoters was studied by DNase I footprinting techniques. These studies revealed an absolute requirement for the initiating nucleotide (GTP) for each phage RNA polymerase to bind specifically to and protect its cognate promoter from DNase I digestion. In the absence of the initiating nucleotide, both enzymes randomly bind DNA with lower affinity. No other nucleotide can substitute for GTP; however, the addition of GTP + ATP, which causes the synthesis of a hexamer RNA (pppGpGpGpApGpA), makes the DNA-RNA-protein complex highly stable. Nitrocellulose filter binding studies confirmed these observations. On the basis of these results we propose that the binding of the initiating nucleotide (in this case, GTP) drives the phage RNA polymerase into an "initiation conformation" in which the random DNA-binding property of the enzyme is converted to a promoter-specific recognition, and the polymerase is primed to initiate transcription.

Bacteriophage lambda↗

PubMed1986

Identification of ribosome-bound eukaryotic initiation factor 2.GDP binary complex as an intermediate in polypeptide chain initiation reaction.

Studies on the formation and release of the eukaryotic initiation factor (eIF)-2.GDP binary complex formed during eIF-5-mediated assembly of an 80 S initiation complex have been carried out. Incubation of a 40 S initiation complex with eIF-5, in the presence or absence of 60 S ribosomal subunits at 25 degrees C, causes rapid and quantitative hydrolysis of ribosome-bound GTP to form an eIF-2.GDP binary complex and Pi. Analysis of both reaction products by Sephadex G-200 gel filtration reveals that while Pi is released from ribosomes, the eIF-2.GDP complex remains bound to the ribosomal initiation complex. The eIF-2.GDP binary complex can however be released from ribosome by subjecting the eIF-5-catalyzed reaction products to either longer periods of incubation at 37 degrees C or sucrose gradient centrifugation. Furthermore, addition of a high molar excess of isolated eIF-2.GDP binary complex to a 40 S initiation reaction mixture does not cause exchange of ribosome-bound eIF-2.GDP complex formed by eIF-5-catalyzed hydrolysis of GTP. These results indicate that eIF-2.GDP complex is directly formed on the surface of ribosomes following hydrolysis of GTP bound to a 40 S initiation complex, and that ribosome-bound eIF-2 X GDP complex is an intermediate in polypeptide chain initiation reaction.

Animals↗

PubMed1985

Purification and characterization of a guanosine diphosphatase activity from calf liver microsomal salt wash proteins.

A potent guanosine diphosphatase activity that hydrolyzes GDP to 5'-GMP + Pi has been isolated and purified from the salt wash proteins of calf liver microsomes. The purified enzyme, a monomeric protein of approximate Mr 46,000, possesses nucleotide substrate specificity since, among the nucleoside diphosphates and triphosphates tested, only GDP and UDP are hydrolyzed by the enzyme. The relative affinity of the enzyme for GDP is, however, much higher than for UDP. The effect of the enzyme on the binary complex formed between eukaryotic initiation factor 2 (eIF-2) and GDP has also been investigated. The enzyme neither hydrolyzes GDP bound to eIF-2 nor catalyzes the exchange of eIF-2-bound GDP with GTP even in the presence of Met-tRNAf. The enzyme, therefore, is presumably not involved in recycling of eIF-2 in eukaryotic polypeptide chain initiation reaction. The possible biological function of the enzyme in maintaining the cellular pool of GTP-GDP is discussed.

Animals↗

PubMed1985

Eukaryotic initiation factor 5 from calf liver is a single polypeptide chain protein of Mr = 62,000.

Eukaryotic initiation factor 5 (eIF-5), which specifically catalyzes the joining of a 60 S ribosomal subunit to a 40 S initiation complex to form a functional 80 S initiation complex, has been purified from ribosomal salt wash proteins of calf liver. The purified factor exhibits only one polypeptide band of Mr = 62,000 following electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. The native protein has a sedimentation coefficient of 4.2 S and a Stokes radius of 33 A which is consistent with eIF-5 being a monomeric protein of Mr = 58,000-62,000. Less pure preparations of eIF-5 elute in gel filtration columns with an apparent Mr of 160,000-180,000 presumably due to association of eIF-5 with other high molecular weight proteins since eIF-5 activity present in such preparations can also be shown by gel electrophoretic separation under denaturing conditions to be associated with a 62,000-dalton protein. Furthermore, eIF-5 purified from calf liver extracts with or without a number of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity of purified preparations. The purified factor catalyzes the hydrolysis of GTP present in 40 S initiation complexes in the absence of 60 S ribosomal subunits. The presence of 60 S ribosomal subunits neither stimulates nor inhibits the hydrolysis of GTP. However, the factor cannot mediate 40 S or 40 + 60 S ribosome-dependent hydrolysis of GTP in the absence of Met-tRNAf or other components required for 40 S initiation complex formation. It can be calculated that 1 pmol of eIF-5 protein can catalyze the formation of at least 10 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.

Animals↗

PubMed1985

Formation and release of eukaryotic initiation factor 2 X GDP complex during eukaryotic ribosomal polypeptide chain initiation complex formation.

The formation and release of an eukaryotic initiation factor (eIF)-2 X GDP binary complex during eIF-5-mediated assembly of an 80 S ribosomal polypeptide chain initiation complex have been studied by sucrose gradient centrifugation analysis. Isolated 40 S initiation complex reacts with eIF-5 and 60 S ribosomal subunits to form an 80 S ribosomal initiation complex with concomitant hydrolysis of an equimolar amount of bound GTP to GDP and Pi. Sucrose gradient analysis of reaction products revealed that GDP was released from ribosomes as an eIF-2 X GDP complex. Evidence is presented that eIF-5-mediated hydrolysis releases the GTP bound to the 40 S initiation complex as an intact eIF-2 X GDP complex rather than as free GDP and eIF-2 which subsequently recombine to form the binary complex. Furthermore, formation and release of eIF-2 X GDP from the ribosomal complex do not require concomitant formation of an 80 S initiation complex since both reactions occur efficiently when the 40 S initiation complex reacts with eIF-5 in the absence of 60 S ribosomal subunits. These results, along with the observation that the 40 S initiation complex formed with the nonhydrolyzable analogue of GTP, 5'-guanylylmethylene diphosphonate, can neither join a 60 S ribosomal subunit nor releases ribosome-bound eIF-2, suggest that following eIF-5-mediated hydrolysis of GTP bound to the 40 S initiation complex, both Pi and eIF-2 X GDP complex are released from ribosomes prior to the joining of 60 S ribosomal subunits to the 40 S initiation complex.

Animals↗

PubMed1985

Nucleotide sequence of a major class-III phage-T3 RNA-polymerase promoter located at 98.0% of phage-T3 genetic map.

The entire nucleotide sequence of a 409-bp HincII fragment, located within the MboI-E fragment on bacteriophage T3 DNA and containing a major class-III T3 RNA polymerase promoter positioned at 98% on the standard T3 genetic map, has been determined. Alignment of this class-III promoter with previously determined T3 RNA polymerase promoters, with start points of transcription (+1) in register, indicates high degree of sequence conservation between position -16 to +6 among all T3 RNA polymerase promoters. The conserved portion of the (-) strand sequence is 5'-A-TA-T-AT-A-C-C-C-T-C-A-C-T-A-A-A-G-G-G-A---3'. This fragment also contains an open reading frame (ORF) with a translational start codon located at position +146 which is preceded by a potential ribosome binding site (RBS). There is more than 70% amino acid-sequence homology between the deduced sequences of the -NH2 terminal region of this putative T3 phage protein and the corresponding protein coded by bacteriophage T7 (protein of T7 gene 19.5).

Base Sequence↗