今日推送的文章是发表在Journal of Biological Chemistry上的“Functional domains of a ribosome arresting peptide are affected by surrounding nonconserved residues ”，通讯作者为美国阿拉巴马州亨茨维尔亨茨维尔阿拉巴马大学的Heather N. G. Judd。
Expression of the Escherichia coli tnaCAB operon responsible for L-tryptophan (L-Trp) transport and catabolism is regulated by L-Trp-directed translational arrest and ribosomal blocking peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide that are involved in the formation of L-Trp binding sites at ribosomal exit channels and inhibit ribosomal function. The aim of this article is to understand whether non-conserved amino acids surrounding these key conserved residues play a functional role in TnaC-mediated ribosomal stagnation.
The authors investigated the effects of mutations within two genes that partially restored L-Trp-dependent induction in LOF mutants of TnaC RAP (Tables 1 and 2). The ligated reporter construct P24 mutation was used (Table 2), while R23H could partially inhibit almost all LOF mutations tested at D16, W12, and P24, with the exception of W12A (Table 2). The authors showed that nascent TnaC(S10P) peptides induced the accumulation of ribosomal arrest at lower L-Trp concentrations than WT TnaC peptides (Figure 3) and that S10P had no general effect on action compared to R23F. puromycin in vitro, but conversely increases the ability of L-TRP to block puromycin (Figure 4). In addition, nascent TnaC peptides containing S10P do not affect the activity of PTCs in TnaC-blocking ribosomal complexes in the manner of TnaC(R23F) peptides. It also supports the need for two distinct domains called sensor and stall domains for TnaC peptide induction of ribosomal arrest, with S10P influencing the sensor domain and R23F influencing the stall domain (Figure 5). The sensor domain used to bind free L-Trp within the channel contains conserved TnaC residues W12 and D16 and, to a lesser extent, adjacent less conserved residues. The arrest domain, which consists of P24 and adjacent residues (e.g., R23), acts on PTC to reduce the binding kinetics of active molecules (e.g., puromycin and RF2) at ribosomal active sites.
Figure 3
Figure 4
Figure 5
Three TnaC mutations, S10P, R23H, and R23F, have been identified to increase TnaC sensitivity to L-Trp. R23F was first identified in TnaC peptides, where it can block ribosomes in the elongation phase, while S10P and R23H mutations were isolated as part of this work as inhibitory mutations, resuming induction of non-inducible D16E peptides. The R23F mutation yields a stable ribosomal arrest complex for obtaining a high-resolution 2.4 Å cryo-EM model of TnaC-blocked ribosomes bound by L-Trp. Changes in R23 TnaC residues reduced the sensitivity of TnaC to puromycin transfer, independent of L-Trp (Figure 4D). Since the R23 amino acid residue is in close proximity to the PTC, the authors anticipate that changes in this position can reduce the kinetics of action of puromycin and RF2 on the PTC, allowing the TnaC-ribosomal complex to interact with L-Trp with higher efficiency (Figure 1). This may explain the inhibitory effect of R23H on LOF P24C mutations (Table 2). As previously proposed, the authors suggest that the presence of His or Phe residues at the R23 site conflicts with the Q252 residue of RF2 in the PTC A site and reduces the fitness rate of RF2. However, the possibility that the R23H/P24C double mutation may also affect Rho binding cannot be ruled out, as previously suggested.
Figure 1
The current TnaC-ribosomal structure shows that the S10 residue near the L-Trp binding site is just outside the contractile region. Therefore, a change in the S10P residue is unlikely to physically hinder the proper regulation of the A-site molecule compared to a change in R23F/H and therefore must exert its forces in a different way. The authors have demonstrated that S10P increases L-Trp sensitivity without affecting PTC (Figure 4) and hypothesizes that the presence of Pro residues at TnaC position 10 increases the affinity of the L-Trp binding site. Based on the density map obtained using the 2.4 Å cryo-EM structure, no interaction between the S10 residue and the free L-Trp, uL22 protein, or 23S rRNA nucleotides was observed. In addition, S10 is not a conserved residue in the TnaC peptide family. However, experiments using N-terminal truncated TnaC peptides have shown that the W12 residue should be preceded by at least two residues to maintain a functional peptide. This observation suggests that W12 is dependent on the spatial distribution of S10 residues within the ribosomal exit channel, and that the presence of proline residues at this location may increase the chances of W12 establishing a functional conformation (Figure 5). Replacing W12 residues with small amino acids (e.g., Ala) decreases the ability of S10P to inhibit the LOF phenotype (Table 2), suggesting that the increased affinity for binding L-Trp depends on the size of the 12th residue. According to the authors, this suggests that, as observed by cryo-EM, the formation of binding sites capable of accommodating free LTrp requires long chains or bulky amino acid residues at the 12 position of TnaC. In addition, once TnaC interacts with free L-Trp, the large amino acid at position 12 of TnaC may also facilitate the transmission of structural information to PTC via TnaC peptides. These last ideas are supported by the lack of inhibitory activity observed in R23H changes. W12A mutations (Table 2). That is, although the R23H mutation reduces the activity of PTCs, the TnaC(R23H) peptide must still rely on the binding of free L-Trp for maximum blocking capacity (Figure 5).
Contrary to the results obtained with W12 changes, the data suggest that long-chain amino acid residues at the D16 locus (e.g., Lys) impede the ability of S10P or R23H mutations to inhibit the LOF phenotype (Table 2). The D16 residue interacts with the U2609 23S rRNA nucleotide residue at the peptide exit channel and is important for L-Trp induction of the tRNA operon. D16 is located in the region between the two α helices of the TnaC peptide, forming a 110° hinge that allows the formation of a TnaC arrest configuration. D16 is important for the role of other WT TnaC peptides (Table 2). However, residue identity at position 16 becomes less important when TnaC peptides contain S10P or R23H, as small residues other than Asp (e.g., Ala) allow TnaC (S10P or R23H) to function (Table 2). The small size of the Ala residue at position 16 can be used as a placeholder within the peptide for proper localization of the essential rRNA residue of the channel contained in the sensor or stall functional domain. Structural changes in TnaC peptides produced by S10P and R23H may shift the position of the D16 residue within the exit channel closer to the 23S rRNA residue. Replacing the 16th position of TnaC with small amino acid residues may still contribute to the formation of the hinges required for the TnaC arrest configuration.
The work presented here shows how mutations in non-conserved residues can be exploited to modulate RAP responsiveness. Both S10P and R23H mutations increased the sensitivity of TnaC to detect L-Trp and partially inhibited the LOF phenotype associated with some, but not all, abolition-induced mutations. The authors suggest that both S10P and R23H inhibitors alter the overall structure of the TnaC peptide, eliminating the need to fold into the essential residues required for functional configurations. These data support the idea that non-conserved residues can also significantly modulate the function of this inhibitory peptide. The authors' observations of the versatility of TnaC peptide variants in inducing ribosomal arrest suggest that sequences of regulatory arrest peptides from different bacteria have been evolutionarily selected to adjust the expression of tna operon based on L-Trp levels in the environment. Future biochemical and structural studies may determine whether TnaC-regulated tna operons from other bacterial strains respond to specific L-Trp concentrations, and whether these TnaC peptide variants can sense other metabolites, which may shed light on how rationally designed.