Translational control of ribosomal protein L10 synthesis occurs prior to formation of first peptide bond

Abstract

A simplified DNA-directed in vitro system has been developed to study the regulation of the synthesis of ribosomal protein L1O by measuring the formation of the first dipeptide, fMet-Ala. The results show that the inhibition of L1O syn. thesis by L1O (autoregulation) occurs at or prior to the formation of the first peptide bond.It is now known that the synthesis ofEscherichia coli ribosomal proteins is under both transcriptional and translational control. At the level of transcription, ribosomal protein synthesis is under stringent control mediated by the unique nucleotide guanosine-3'-diphosphate-5'-diphosphate (ppGpp) (1). More recently, it has been shown that the synthesis ofcertain ribosomal proteins can be autoregulated at the level of translation. In this process, specific ribosomal proteins inhibit their own synthesis and, in some cases, the synthesis of other ribosomal proteins whose genes are on the same operon. Thus, ribosomal proteins S4 (2, 3), S7 (4), S8 (2), Li (2, 3, 5), LA (2, 6), and L10 (7-10) have been shown to be autoregulators, functioning at the level of translation.Our laboratory has recently studied the effect of L10 on its own synthesis in DNA-and mRNA-directed in vitro systems (7). Although considerable progress has been made in using defined components in these in vitro systems (11,12), their complexity to some extent limits their usefulness in studying the mechanism of autoregulation.The present study describes a simplified in vitro system that can be used to study the regulation of gene expression at transcription or translation initiation by measuring the formation of NH2-terminal small peptides characteristic ofthe gene product. Evidence is presented that the inhibition by L10 ofits own synthesis occurs at, or prior to, the formation of the first peptide bond of L10. MATERIALS AND METHODSE. coli containing Arif18 phage was obtained from J. B. Kirschbaum (Harvard University, Cambridge, MA). E. coli JF943 containing either plasmid pNF1337 or 1341 were kindly supplied by J. Friesen (York University, Ontario, Canada). Ribosomal protein L12 was purified as described (13) of Ala-tRNA and Ser-tRNA synthetase (14). N10-Formyl-H4-folate-Met-tRNA transformylase was purified by a described procedure (15). The acylation and transformylation reactions were carried out as described (15)(16)(17)(18). At the end of the incubations, the reaction mixtures were extracted with phenol and the acylated tRNA species were precipitated with alcohol and dialyzed overnight against 2 mM K acetate (pH 5.5).For Ala-and Ser-tRNA synthesis, unfractionated E. coli tRNA was used; purified tRNAP et was used to prepare fMet-and L-methionine-L-serine were purchased from Sigma. N-Formylmethionylserine was synthesized as follows (19). To 71 mg of L-methionyl-L-serine dissolved in 1.26 ml of 98% formic acid was added dropwise 0.42 ml of acetic anhydride at 10°C. The mixture was stirred for 1 hr at room temperature, and 0.5 ml of ice-water was added. The mixture was brought to dryness under vacuum and the white p...

In vitro synthesis of the tryptophan operon leader peptides of Escherichia coli, Serratia marcescens, and Salmonella typhimurium.

“…Similarly, the S. marcescens peptide was labeled by tryptophan, arginine, histidine, and leucine but not by proline or lysine (Fig. 2B) (17)(18)(19). Plasmid pAD7 and its HindIII fragment, both of which contain the coding region for the S. typhimurium trp leader peptide, direct the synthesis of the peptides fMet-Ala and fMet-Ala-Ala, the products predicted from the DNA sequence ( Fig.…”

“…We also describe experiments with a highly defined system for di-and tripeptide synthesis (17)(18)(19) Plasmids. DNA containing the E. coli trp promoter-operator and leader peptide-encoding region was isolated as a 360-basepair (bp) Sau3A-Hae III restriction fragment from plasmid pGM22.…”

“…Formation of one or the other of the alternative regulatory secondary structures is believed to be determined by the position of the ribosome engaged in translation of the short coding region located early in trp leader RNA. In the amino acid biosynthetic operons of the bacterial species that have been studied to date, the peptides specified by the short coding regions are [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] residues in length and are rich in the amino acid(s) that controls the operon. One essential feature of the above model is a requirement for efficient synthesis of the short peptide encoded in the leader region of the respective operons.…”

“…The reaction mixture contained DNA template, purified RNA polymerase, initiation factors, elongation factor(s), ribosomes, charged tRNAs, salt, and other components. Analysis of the dipeptide fMet-Ala used an ethyl acetate extraction procedure (19), whereas TLC was used to assay for the tripeptide fMet-Ala-Ala (17,18). Linear DNA fragments were used as templates in the purified peptide-synthesizing system.…”

“…DNA-dependent di-and tripeptide synthesis was performed as described (17)(18)(19). The reaction mixture contained DNA template, purified RNA polymerase, initiation factors, elongation factor(s), ribosomes, charged tRNAs, salt, and other components.…”