fMet Research Articles

Gag-Related polyproteins of Moloney murine leukemia virus: evidence for independent synthesis of glycosylated and unglycosylated forms.

Immunoprecipitation of labeled extracts from murine leukemia virus-infected cells with antisera specific for internal structural (gag) proteins yields three major gag-related polyproteins with molecular weights of 180,000 (Pr180gag-pol), 80,000, and 65,000 (Pr65gag). It has been shown by others that Pr65gag is the immediate precursor of the internal structural (gag) protein, and that Pr180gag-pol is the precursor to reverse transcriptase. In studies reported here, the 80,000-dalton gag-related polyprotein from Moloney strain murine leukemia virus (M-MuLV)-infected cells was found to be glycosylated by the following criteria: (i) incorporation of [3H]mannose, (ii) a change in electrophoretic mobility upon digestion with endoglycosidase H, and (iii) a change in electrophoretic mobility when glycosylation was inhibited by treatment of the cells with tunicamycin during labeling. The 80,000-dalton gag polyprotein has therefore been designated GpP80gag. The unglycosylated form of GpP80gag was a polypeptide of 75,000 daltons. A comparison of [3H]mannose and [3H]galactose labeling experiments suggested that GpP80gag is further glycosylated to yield a glycopolypeptide of 95,000 daltons. This 95,000-dalton polypeptide is relatively rapidly cleaved to yield two glycopeptides of 55,000 and 40,000 daltons which are released into the cell culture fluid, as soluble proteins. Cell-free translation of M-MuLV genomic RNA resulted in two major gag-related products of 75,000 and 65,000 daltons. The 65,000-dalton gag-related cell-free translation product comigrated with Pr65gag, and the 75,000-dalton cell-free product comigrated with the unglycosylated form of GpP80gag. Both of the gag-related cell-free translation products could be labeled with [35S]formyl methionine, which is incorporated only as the N-terminal amino acid during translation. Other investigators have shown that GpP80gag and Pr65gag differ at their N-termini, and these results combined with those reported here suggest that GpP80gag and Pr65gag are translated from two separate initiation sites in M-MuLV RNA.

Polyoma virus has three late mRNA's: one for each virion protein

Polyoma virus mRNA, isolated from the cytoplasm of 3T6 cells late after infection and purified by hybridization to HpaII fragment 3 of polyoma virus DNA, was separated on 50% formamide-containing sucrose density gradients, and the fractionated RNA was recovered and translated in vitro. Analysis of the cell-free products showed that the minor virion protein VP3 was synthesized from an mRNA sedimenting at approximately 18S betweeen the 19S VP2 mRN and the 16S VP1 mRNA. Other experiments showed that the VP2 and VP3 can be labeled with formyl methionine from initiator tRNA. We conclude that there are three late polyoma virus mRNA's, each directing the synthesis of only one viral capsid protein.

Catalysis of the peptide bond formation by 50 S subunits of E. coli ribosomes with N-(formyl) methionine ester of adenylic acid as peptide donor.

50 S subunits of E. coli ribosomes catalyze the reaction of the 2'(3')-N-(formyl) methionine ester of adenosine 5'-phosphate and Phe-tRNA resulting in peptide bond synthesis. Cytidine 5'-phosphate stimulates this process on 50 S ribosomal subunits as well as on intact ribosomes. The obtained data show that the areas of the peptidyltransferase donor site which binds the 3'-terminal fragment of peptidyl-tRNA possess completely formed structures on 50 S ribosomal subunits.

Specific binding of formylated initiator-tRNA to Escherichia coli RNA polymerase.

E. coli fMet-tRNAfMEet and E. coli RNA plymerase (RNA nucleotidyltransferase; EC 2.7.7.6; nucleoside-triphosphate:RNA nucleotidyltransferase) form a 1:1 complex with an apparent association constant of 9.0 X 10(6)M-1 at 37 degrees. The affinity of polymerase to tRNA depends on the tRNA as well as the formyl methionine moiety. Core polymerase has a greatly reduced affinity for initiator tRNA. Optimal binding conditions are similar to those that are also optimal for binding initiator tRNA to ribosomes. Binding of initiator tRNA to polymerase stimulates the transcription of lambda plac DNA, as determined in a crude cell-free system for beta-galactosidase (EC 3.2.1.23; beta-D-galactoside galactohydrolase) synthesis as well as in a highly purified transcription system.

Initiation of phi-X-174 RF-primed protein with N-formylmethionine.

Initiation of phi-X-174 RF-primed protein with N-formylmethionine.

Contradictions in LSD research.

Contradictions in LSD research.

Formyl Methionine tRNA and Valine tRNA Available

Formyl Methionine tRNA and Valine tRNA Available

Formyl methionine tRNA and valine tRNA available.

Formyl methionine tRNA and valine tRNA available.

Deformylation of N-formylmethionine by Escherichiacoli extracts

Deformylation of N-formylmethionine by Escherichiacoli extracts