Primary structures of mutationally altered ribosomal protein L7/L12 and their effects on cellular growth and translational accuracy

Abstract

The amino acid sequences of mutationally altered ribosomal protein L7/L12 from four different rplL mutants of Escherichia coli were determined and correlated with some features of the mutant ribosomes. Two of the rplL mutations are deletions around position 40, which give rise to a shortened hinge region between the two domains of L7/L12. The other two mutants harbor point mutations at position 74 (Gly+Asp) or at position 82 (Glu+Lys), which are in or close to an evolutionarily conserved sequence in the C-terminal domain. The two latter mutations are associated with decreased rates of growth and translational elongation. All four mutants show increased nonsense codon read-through in vivo. Ribosomes from one of the deletion mutants show clearly increased missense error rates in vitro.Some mutationally altered ribosomal proteins affect translational accuracy both in vivo and in vitro. The best characterized examples of far are mutants with changed S12 (rpsL), which show a more accurate phenotype, and with altered S4 (rpsD), which are less accurate during translation [l -81. These mutants [9, 101 are primarily defective in translational proof-reading step(s) [ll -151. Recent observations show that S5 (rpsE) mutations, which are associated with a ribosomal ambiguity (Ram) phenotype in vivo and in vitro, produce ribosomes which have a decreased proof-reading intensity [16,17,441. Furthermore, mutations in rpsQ (S17) and in rplF (L6) might give a more accurate translation but such mutants are not so well characterized as those affecting S4 and S12 [18, 191. Recently ribosomal protein L7/L12 was shown to be involved in the maintenance of translational accuracy. It was found that some mutations in the gene coding for this protein (rplL) increase the translational error rates both in vivo and in vitro. Furthermore, the increased error level, characteristic of the mutant ribosomes containing altered forms of L7/L12, can be correlated with a decreased proof-reading efficiency. The increased translational misreading in vivo is reversed by the introduction of an rpsL allele, which gives a Str-R phenotype. Therefore it appears that some mutants with altered L7/L12 in many respects are similar to S4 and S5 mutants [7, 9, 20, 441 in that they both increase error frequencies and suppress streptomycin-dependent phenotypes.In the present investigation we have purified the L7/L12 protein [21] from four different rplL mutants and localized the corresponding alterations by determining their amino acid sequences. In addition, we describe the effects of these mutational alterations on translation rates both in vivo MATERIALS AND METHODS Construction of strainsThe E. coli K12 strains used in this study are listed in Table 1. The rplL159, rplL265 and rplL564 alleles were introduced by PI -mediated cotransduction with argE+ into UY128 and UD132 followed by screening for rifampicin sensitivity (Rif-S) as described elsewhere [20]. Strain VT564 was used as the donor of rplL564.Construction of the rplL518 derivatives UY258 and UY278 was...

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

“…Studies during the 1970s first demonstrated the requirement of L7/L12 for the functions of a wide variety of essential translation factors, including G and non-G proteins, in protein synthesis in vitro (7,8,27,28). Subsequent studies isolated mutants of L7/L12, which altered the fidelity and speed of protein synthesis in vivo (29). Recent in vitro studies have converged on a small cluster of conserved, positively charged or aliphatic residues of L7/L12, which may interact with the G protein factors EF-Tu, EF-G, RF3, and IF2 (9,10,30).…”

The length of the interdomain region of the L7/L12 protein is important for its function

“…The experiments on miscoding show that the protein with the inserted fourteen residues (pPRI2, Table 1) has a lower level of miscoding. The partial deletion of the flexible region of the LT/LI2 also affects the miscoding level [9,10,20].…”

“…Expression of the L12 w.t. gene without induction (1) and after induction by IPTO (2); expression of LI2-pPRI 1 (3), LI 2-pPR12 (4), LI2-pPR28 (5), LI2-pPK37 (6) and LI2-pPR38 (7); markers (Pharmacia) 14, 20, 30, 43, 67, and 94 kDa (8,9); purified proteins LI2-pPRI2 (10) and LI2-pPR38 ill).…”

“…Earlier it was shown that ribosomes with a shortened interdomain region of L7/LI2 decrease the translation rate in vitro [9,20] and the protein with deleted interdomain region is not active [20].…”

“…The depletion of L7/Ll2 from ribosomes inh~.bits protein synthesis and decreases elongation factor activity [3][4][5][6][7]. Two known mutations in the N-terminal part of LT/LI 2 influence the level of misreading [8][9][10]. Structural studies of L7/LI2 have shown that it contains two domains.…”