Expression of the gene for Escherichia coli initiation factor IE-3 in vivo and in vitro.

Expression of protein synthesis initiation factor IF-3 in vivo was studied by measuring its level in exponentially growing cells as a function of gene dosage. A strain haploid for infC, the gene for IF-3, was modified to carry one or two additional infC genes giving diploid and triploid strains. Polyploid strains were achieved by the presence of multicopy plasmids expressing the infC gene. When IF-3 levels were measured by quantitative immunoblotting they were found to be proportional to the gene dosage; the presence of a multicopy plasmid thus causes considerable overproduction of IF-3, enabling large quantities to be purified. When lysates were prepared from freshly grown cells, only IF-3 alpha (the long form) was detected; however when IF-3 was purified from a strain containing a multicopy plasmid which overproduced it, the major product found was IF-3 beta (the short form, lacking six amino acids from the N terminus). The synthesis of the two IF-3 forms was also studied by using a cell-free coupled transcription-translation system dependent on exogenous DNA: the IF-3 gene was found to be very efficiently expressed. IF-3 alpha increased more rapidly than IF-3 beta but following the cessation of protein synthesis IF-3 alpha decreased while IF-3 beta still increased. The results suggest that IF-3 alpha is slowly degraded to the beta form. Addition of non-radioactive IF-3 alpha, up to fivefold molar excess over ribosomes, to the synthesizing system in vitro did not inhibit IF-3 synthesis. Synthesis of IF-3 in vitro appears to be sensitive to guanosine 3'-diphosphate 5'-diphosphate.

[1]  J. Hershey,et al.  A sensitive immunoblotting method for measuring protein synthesis initiation factor levels in lysates of Escherichia coli. , 1981, The Journal of biological chemistry.

[2]  M. Grunberg‐Manago,et al.  Physical localisation and cloning of the structural gene for E. coli initiation factor IF3 from a group of genes concerned with translation. , 1980, Gene.

[3]  M. Nomura,et al.  In vitro expression of Escherichia coli ribosomal protein genes: autogenous inhibition of translation. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[4]  L. Lindahl,et al.  Operon-specific regulation of ribosomal protein synthesis in Escherichia coli. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[5]  J. Chamberlain,et al.  Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. , 1979, Analytical biochemistry.

[6]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[7]  M. Shibuya,et al.  Studies on stringent control in a cell-free system. Regulation by guanosine-5'-diphosphate-3'-diphosphate of the synthesis of elongation factor Tu. , 1979, Journal of biochemistry.

[8]  J. Hershey,et al.  Determination of protein synthesis initiation factor levels in crude lysates of Escherichia coli by a sensitive radioimmune assay. , 1978, Archives of biochemistry and biophysics.

[9]  J. Sutcliffe,et al.  Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. , 1978, Proceedings of the National Academy of Sciences of the United States of America.

[10]  J. Hershey,et al.  Purification and characterization of protein synthesis initiation factors IF1, IF2, and IF3 from Escherichia coli. , 1977, Archives of biochemistry and biophysics.

[11]  A. Subramanian,et al.  Separation of two forms of IF‐3 in Escherichia coli by two‐dimensional gel electrophoresis , 1977, FEBS letters.

[12]  F. Neidhardt,et al.  Chemical measurement of steady-state levels of ten aminoacyl-transfer ribonucleic acid synthetases in Escherichia coli , 1977, Journal of bacteriology.

[13]  W. Schaffner,et al.  A rapid, sensitive, and specific method for the determination of protein in dilute solution. , 1973, Analytical biochemistry.

[14]  K. Low Escherichia coli K-12 F-prime factors, old and new , 1972, Bacteriological reviews.

[15]  D. Clewell,et al.  Nature of Col E1 Plasmid Replication in Escherichia coli in the Presence of Chloramphenicol , 1972, Journal of bacteriology.

[16]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[17]  D. Lawrence,et al.  Level of methionyl-tRNA synthetase in merodiploids of Escherichia coli K12. , 1970, European journal of biochemistry.