L16, a bifunctional ribosomal protein and the enhancing effect of L6 and L11

Abstract

L16 exhibits both peptide bond and transesterification activities when reconstituted into 2 M LiCl core particles. L6 and L11, when reconstituted in a similar manner in the absence of L16, manifest significant transesterification activity. Both L6 and L11 enhance the transesterification activity of L16; L11 being more active than L6 in this respect. However, both L6 and L11 have minimal effect on peptide bond formation when reconstituted with L16 at concentrations more than 2.5 M equivalents. Both L6 and L11 exhibit a differential effect on transesterification.The affinity-labelling agents, like PhCH2S02F, diisopropylfluorophosphate and ethoxyformic anhydride, have been used to explore the role of residues in peptide bond formation and transesterification. It is proposed that the Ser-Phe combination present in L16, L11 and L6 is involved in transesterification in addition to the single histidine in L16. The single histidine in L16 appears to be important in the catalysis of peptide bond formation and transesterification.Peptidyltransferase is a central enzyme on the large (50s) ribosomal subunits, which catalyses peptide bond formation [l, 21 and also may be involved in peptide chain termination Experimental approaches, involving partial reconstitution[5], affinity labelling or chemical modifications [6] , mutants [7, 81 and immunological techniques [9], have been used in attempts to delineate the role of the ribosomal proteins essential for peptide bond formation of peptide chain termination. Partial reconstitution studies indicate that L16, which is completely split off the 50s particles by 1.5 M LiCl, is the only protein which restores activity when added back to the depleted core particles at 2.5 molar excess or higher concentration [lo]. L16 appears to be most active protein involved in many catalytic functions of peptidyltransferase [ll]. It is interesting to note that to date Escherichiu coli mutants lacking L16 have not been reported [ll].L6 and L11 are two other proteins which have been shown to exert a stimulatory effect on L16-mediated peptidyltransferase activity. L6 forms a specific complex with 23s RNA [12] and is considered to be located at the aminoacyl-tRNAbinding site of the peptidyltransferase centre [13] whereas L11 has been implicated as part of the domain surrounding the peptidyltransferase centre [lo]. Using an Lll-deficient mutant, L11 has been shown to have little effect on peptidyltransferase activity [7, 81. Antibodies specific to L16 and L11 affected the peptidyl-tRNA hydrolysis and, therefore, were considered to be involved in peptide chain termination [ 141. However, peptidyltransferase and peptide chain termination were little affected in L11-depleted reconstituted particles [15]. The specific role of L11 has recently 13~41.

Reconstruction of peptidyltransferase activity on 50S and 70S ribosomal particles by peptide fragments of protein L16

“…The puromycin and magnesium ion concentrations were 0.25 mM and 5 mM, respectively L11 was able to enhance this activity from 15% to 27% (Table 3). A similar increase in the transesterification activity was observed with native L16 [8]. L11 in conjunction with L6 reconstructed 31 % of the activity (Table 3).…”

“…However, this requirement is alleviated by carrying out the reconstitution in the presence of a 3 -6-fold molar excess of L16 or L16 peptide [8,231. Under the latter conditions, we have previously shown that L6 and L11 preferentially stimulate the transesterification reaction over peptide-bond synthesis [8]. L11 has also been implicated in the termination reaction, presumably scored by transesterification, as being important for the proper functioning of release factors RF-1 and KF-2 [25].…”

“…Ester-bond formation was studied as above but substituting hydroxypuromycin (I .9 mM) for puromycin [8]. EF-P was assayed by its ability to stimulate f[35S]Metpuromycin (or f[35S]Met-hydroxypuromycin) synthesis from ribosome .…”

“…Although other ribosomal proteins (e.g. L2 and L4) also contain such potentially active residues [2], the unique histidine of L16 has to date remained a prime candidate for providing, at least in part, the catalytic activity of the peptidebond formation and transesterification processes [8].…”

“…Although other ribosomal proteins (e.g. L2 and L4) also contain such potentially active residues [2], the unique histidine of L16 has to date remained a prime candidate for providing, at least in part, the catalytic activity of the peptidebond formation and transesterification processes [8]. In this connection, we report here on the reconstruction of both peptidyltransferase activities of 50s and 70s ribosomes by proteolytic fragments of L16 and study the effect of L6, L11 and L15 and the stimulatory protein EF-P on these reactions.…”