Mapping of ribosomal protein genes by in vitro protein synthesis using DNA fragments of lambda fus3 transducing phage DNA as templates

Abstract

hfus3 (or hfus2) transducing phage stimulates the synthesis of 27 ribosomal proteins, elongation factors EF-Tu and EF-G, and RNA polymerase subunit (Y in ultraviolet-irradiated Escherichia roli cells (see a preceding paper, Jaskunas, S. R., Fallon, A. M., and Nomura M. (1977) .I. Bid. Chem. 252, 7323-7336). The presence of the structural genes for these proteins on the hfus3 genome was proven by demonstrating the synthesis of these proteins in a DNA-dependent protein-synthesizing system using Afus3 DNA, or its restriction enzyme fragments, or both, as template. Proteins synthesized in the system were identified by a combination of techniques. including gel electrophoresis, immunochemical reactions, and tryptic peptide analysis. Restriction endonuclease-sensitive sites on kfus3 DNA were mapped, and genes were located using restriction endonuclease fragments of Afus3 DNA, isolated directly or cloned in phage or plasmid vectors, as templates for protein synthesis. Additional information about the locations of the genes was obtained using DNA from insertion and deletion mutants of hfux3. The results obtained in these in oilro experiments (rf. Fig. 2), and those obtained in the in uiuo experiments from preceding papers in this series, are in excellent agreement and map the essential genes on Afun and their promoters as follows (going counter-clockwise on the E. coli chromosome): R,,.. (S12, S7), EF-G, EF-Tu; k,,), SlO, LB, (1~4. L2. L23), (L22. S19), s3, (S17, L16, L29); hUC, L14, L34, L5, S14, S8, L6, LlR, S5, (130, L15); pa, S13, Sll, S4, IY, L17. Re-examination of the results of previous physiological experiments (Dennis, P. P. (1974) J. Mol. Biol. 89, 223- 232) using the present information on r-protein gene organization demonstrates that the increase in the differential synthesis rate of ribosomal proteins during the transition period following the nutritional shift-up is primarily due to the increase in the frequency of initiation of the transcription of ribosomal protein genes.