In vitro synthesis of the first dipeptide of the beta subunit of Escherichia coli RNA polymerase.

Abstract

Plasmids pNF1337 and pNF1341, which contain part of the LIO operon including the RNA polymerase 13-subunit gene, have been used as templates in vitro to investigate expression of the p-subunit gene. For these studies, the synthesis of the first dipeptide of the 13 subunit, fMet-Val, was measured instead of that of the entire protein. By using this dipeptide system, we studied the effects of RNA polymerase holoenzyme and L factor (nus A-gene product) on fMet-Val synthesis and compared the relative effects of the primary and secondary promoters in the L1O operon on expression of the 13-subunit gene. The results show that the inhibitory effect of RNA polymerase on P-subunit synthesis and the stimulatory effect of L factor occur before formation of the first dipeptide bond. In this in -vitro system, the secondary promoters account for about 50% of the total fMet-Val synthesized. Although the primary promoter is sensitive to guanosmne 5'-diphosphate 3'-diphosphate in-vitro, the secondary promoters are not affected by this nucleotide.The DNA coding for the /3 subunit ofRNA polymerase is part of a polycistronic operon that also contains the genes for ribosomal proteins L10 and L12 and the /3' subunit of RNA polymerase (1-3). A major promoter for all of these genes is located about 380 base pairs upstream from the L10 gene (1, 4, 5), although evidence for secondary promoters (5-10) that could influence the expression of the L12 and /3-andf/'-subunit genes has been obtained. This operon has received special attention because of the unique regulation that controls the expression of these gene products. In previous studies in vitro, the ratio of synthesis of the four products, L1O/L12//3 subunit/,B' subunit, was about 1:4: <0.1 : <0.1 (11). It is still not entirely clear how the synthesis of these proteins is regulated, but both transcriptional and translational controls are involved. In addition to the secondary promoters, it has been demonstrated in vitro that (i) L10 inhibits its own synthesis (autoregulation) at the level of translation (12, 13); (ii) RNA polymerase inhibits ,B and /3'-subunit synthesis (11,(14)(15)(16); (iii) a transcriptional attenuator site is present in the intracistronic region between L12 and the P subunit (6, 17); and (iv) L factor (18), which is the nus A gene product (19), stimulates /3-subunit synthesis (11).We previously studied the DNA-dependent in vitro synthesis ofthe /3 subunit, L10, and L12 by using a crude protein-synthesizing system (7,8,20) as well as a highly defined system containing more than 30 purified factors (11). In those earlier studies, DNA from the transducing phage Arifdl8 or from the plasmids pNF1337 and pNF1341 was used as template. Using pNF1337, we recently developed a simplified in vitro system to study the regulation of L1O synthesis (21). In this procedure, formation of the first dipeptide (fMet-Ala in the case of L10) was measured in lieu ofthat ofthe entire protein. In the-present study, this in vitro system was used to investigate the synthesis of the /...

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.…”

“…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.…”

“…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.…”

“…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.…”