Why does srp stop translation

Pyruvate kinase from rabbit muscle was purchased from Roche, and proteinase K from Tritirachium album from Sigma. SRP was formed by incubating 4. Between seven and nine replicates of each experiment were carried out for s.

Replicate traces were averaged prior to global fitting. To accommodate the different starting fluorescence of each average trace which increased linearly with Bpy-SRP concentration , a different fluorescence offset was provided for each fluorescence signal. These offsets were initially fitted, but constrained for the final fit. The kinetic constants and errors reported are the best-fit values and standard errors in the fit, respectively. Gels were imaged using an FLA fluorescence scanner Fujifilm and nm laser excitation.

Bands corresponding to peptides with 50 or more amino acids were identified relative to a Lep50 standard, and quantified with ImageJ free download from NIH. Translation efficiencies were calculated as percent of the intensity at s and averages and standard deviations were computed for each time point based on three or four independent experiments.

At various time points, aliquots containing 2. Digestion was carried out for 10 s prior to quenching in 0. After standing on ice for at least 30 min, precipitated peptides were filtered over 0. These labels do not impair SRP-ribosome interactions as equilibrium titrations yielded K d values around nM comparable with other labels at different positions 12 — Synthesis of leader peptidase Lep , an inner-membrane protein with an N-terminal SAS residues 4—22 that is recognized by SRP, was carried out on donor-labeled ribosomes in a highly efficient in-vitro translation system from E.

Control measurements were performed with donor-only and acceptor-only complexes. The resulting fluorescence time course reveals a rapid fluorescence increase during the first 10 s and an additional increase starting at about 40 s. Preliminary analysis of the time courses by exponential fitting reveals that the rapid phase comprises two kinetic steps.

The donor-only control reveals that the rapid fluorescence increase is predominantly due to a change in donor fluorescence, indicating a change in the environment of the label. The rapid, biphasic increase of the signal is observed independent of mRNA length, suggesting that it represents binding of SRP to translating ribosomes carrying short nascent peptides before the SAS emerges from the polypeptide exit tunnel.

Rapid and slow phases of SRP recruitment to translating ribosomes. Fluorescence traces are offset for clarity, and dotted lines indicate the respective initial fluorescence. The time preceding the FRET increase starting at 40 s likely reflects the minimum time required to synthesize a peptide chain of 45—50 amino acids. These experiments show that nascent chains of 50 amino acids and longer appear after 40 s of translation. Thus, the initial rapid recruitment of SRP to the ribosome is followed by a conformational rearrangement, the rate of which is limited by translation.

Concentration dependence of SRP binding to translating ribosomes. Fluorescence traces are offset for clarity and dotted lines indicate the respective starting fluorescence. C Combined amplitudes of the biphasic fluorescence change during the rapid phase of binding were obtained by double-exponential fitting of time courses up to 10 s, disregarding the delay and the slow fluorescence increase seen with LepRNC.

Hyperbolic fitting yielded K d values for the initial binding complex of — nM. Error margins in C and D represent standard errors of the double-exponential fits. To determine rates of SRP binding and subsequent rearrangements we adopted global fitting using a comprehensive kinetic model. We started by globally fitting traces that showed only the rapid fluorescence change Lep 25, Lep35 and Lep40 , at all SRP concentrations tested 18 traces total; Supplementary Figure S4.

In order to adequately fit this data set, SRP binding had to be modeled as a reversible two-step process with initial binding followed by a conformational change; a one-step model did not produce satisfactory fits Supplementary Figure S4. In order to fit both rapid and slow phases of the time courses it was necessary to incorporate nascent-chain synthesis into the global fitting model. For the short nascent chains, SRP binding is modeled by a two-step process as described above.

To account for the increase in affinity of the SRP—RNC complex upon growth of the nascent chain from 40 to 50 amino acids, we have introduced an additional parameter, k -2,long. That study showed that the high affinity of SRP binding to the LepRNC, compared to non-translating ribosomes, results from a lower reverse rate of a conformational rearrangement.

Furthermore, the rate of translation characterized by k trans , the average number of amino acids incorporated into peptide per second, is assumed to be the same for all RNCs regardless of nascent chain length. Because the rate of translation in the E. Translation is modeled left to right as a conversion of initiation complex IC into LepRNC in a series of irreversible steps with six intermediates.

A switch from low to high SRP affinity is modeled upon transition from Lep40 to Lep50 by a reduced rate of the reverse conformational rearrangement k -2 see text. To provide direct information about the rate of translation, and thus improve the fit of k trans , the average of four independent translation time courses is also included in global fitting Supplementary Figure S3B.

Because the affinity of SRP for binding to RNCs carrying long nascent chains is very high, we used additional experimental data to constrain the parameter k -2,long. The fits, shown as red lines along with the corresponding traces, describe the observed traces with high accuracy, despite the complexity of the signal changes. After incorporation of 50 amino acids, the RNC—SRP affinity is increased substantially, as k -2 decreases fold, from 0.

Previous crosslinking data suggested that a Lep nascent chain of about 40 amino acids can reach SRP bound near the exit site as well as the SecY translocon In order to directly test when the N terminus of the nascent Lep peptide emerges from the ribosome in our in-vitro experiments, we probed the proteinase sensitivity of the N-terminal fMet of Lep in a co-translational assay.

The amount of f[ 3 H]Met precipitated initially increases, presumably reflecting the increasing efficiency of TCA precipitation of longer peptides. However, at 30 s the f[ 3 H]Met recovery starts to decrease, indicating that the N terminus of the nascent chain becomes accessible for PK due to exposure outside the peptide exit tunnel. Delay between emergence of the nascent chain and SRP rearrangement. A Nascent-chain emergence from the peptide exit tunnel monitored by PK cleavage of the N terminus.

After quenching, the Lep peptide was released by alkaline hydrolysis, precipitated with TCA, collected on nitrocellulose filters, and quantified by counting 3 H Materials and Methods. The data from A are inverted and normalized closed circles and are plotted along with the normalized fluorescence change from co-translational recruitment of SRP to the RNC monitored by FRET gray trace.

Thus, the rearrangement takes place when at least 10 amino acids have emerged from the peptide exit tunnel of the ribosome, seven of which belong to the Lep SAS, which in total comprises 19 amino acids. This result indicates that SRP can bind to a partially exposed hydrophobic signal-anchor sequence. The present observation of SRP binding to translating ribosomes carrying short nascent chains is at variance with results obtained by single-molecule FRET measurements employing other fluorescence labels at different positions Rather, the SRP—RNC complex was apparently only visible after emergence of the SAS, prompting the suggestion that emergence of the nascent chain causes an increase in the rate of association.

Another potential explanation for the discrepancy is provided by the observation that polyamines suppress the fluorescence change related to SAS-independent SRP binding Supplementary Figure S6. Fitting those data by two different approaches suggests that the rate of the SAS-independent phase changes with the SRP concentration in a hyperbolic manner, consistent with a bimolecular binding event followed by a rearrangement.

The global rate of SAS-dependent recruitment has a residual concentration dependence owing to the contribution of the first SAS-independent phase Supplementary Figure S6 , rather than an increased bimolecular association rate constant Previous equilibrium and pre-steady-state kinetic analyses identified an early, SAS-independent recruitment of SRP to non-translating ribosomes or stalled RNCs carrying short nascent chains. Here, we show that early recruitment occurs with actively translating ribosomes as well.

The formation of the early complex, which is of moderate affinity, is independent of the presence or exposure of an SAS. It proceeds as a rapid two-step reaction, i. The two phases for SRP binding observed here correspond to steps 2 and 3 identified in the previous study performed with stalled LepRNCs A binding step equivalent to the bimolecular step 1 of the previous study is not observed in the present work.

Presumably the higher temperature used here accelerates binding such that the present time resolution is insufficient to resolve binding as a separate step. The transition to a high-affinity complex in the actively translating system occurs with longer nascent-chain lengths compared to what was observed previously with stalled RNCs.

With stalled RNCs 32—35 amino acids of Lep or even nascent chains of 35 amino acids lacking a signal sequence were sufficient to reach the maximum SRP-binding affinity The present data indicate that at conditions of ongoing translation the stabilization of the RNC—SRP complex occurs after translation of about 50 codons. This is consistent with recent single-molecule fluorescence and ribosome profiling studies which suggest that RNCs are most likely bound by SRP when the N terminus of the transmembrane domain is located 40—55 amino acids away from the peptidyl transferase center of the ribosome 17 , Thus, the protein biotinylation can be used for protein targeting assay.

However, in contrast to the prediction of proteomic analysis, the FtsQ targeting showed a slight defect in both the SRP — and MY strains Figure 4C , suggesting that the suppressor mutation played little role in the targeting of FtsQ. These results indicated that the SRP suppressor partially contributed to inner membrane protein targeting and allowed for targeting of some SRP-dependent proteins without causing a failure of targeting of SRP-independent proteins.

Additionally, the expression of heat shock response related chaperones and proteases was not upregulated Supplementary Figure 5B and Supplementary Data Set 1B , suggesting that the heat shock response played little role in compensating the loss of SRP, which is consistent with our previous study Zhao et al.

This suggested that the component of the Sec translocon SecF may not be involved in the protein targeting process without SRP. In contrast, the protein abundance of SecY and FtsY in MY was two times higher than that in the SRP — strain, which is likely caused by the effective targeting of inner membrane proteins with the assistance of translational control.

Overall, protein transport components were unlikely to play a major role in mediating SRP-dependent protein targeting in the absence of SRP. Co-translational protein targeting by SRP is an essential and conserved pathway that delivers most inner membrane proteins to their correct subcellular destinations Saraogi and Shan, Our previous work revealed that SRP was not essential in E.

Isolation of suppressors is a useful strategy to provide insight into certain molecular mechanisms by suggesting which cellular component is involved in an inefficient process Lee and Beckwith, The SRP suppressors involved in protein translation initiation have been identified before, and these suppressors affect the translation process Zhao et al.

In this study, we obtained an SRP suppressor associated with protein translation too. The regulation of translation may be a general way to mediate the translocation of SRP-dependent proteins in the absence of SRP. We observed that in suppressor cells, the ribosomal protein expression was upregulated Figure 2B and the 30S and 50S ribosomal subunits accumulated Figure 3A , but the content 70S ribosome complex was not markedly changed relative to those in the wild-type strain Figure 3A.

This led us to propose that the increased ribosomes are inactive and accumulate in the cytosol. Thus, the SRP suppressor and cellular stress responses may play an important role in ribosomal protein synthesis.

In suppressor cells, the protein translation initiation was impeded Figure 3F , but the initiation time of translation was constant in wild-type and suppressor cells under different growth rates Supplementary Table 3 , which suggested that the pausing at the start of the initiation can be negligible, and the process of 70S ribosome complex entry into the elongation cycle is slower in suppressor cells.

Thus, the SRP suppressor may be associated with the transition from initiation to elongation. The closely related relationship between translation initiation and elongation Riba et al. Furthermore, we showed that the translation fidelity was decreased in suppressor cells Figures 3D,G.

Because the fidelity of translation initiation is modulated by the initiation factors Ayyub et al. We observed that the fidelity of translation elongation was also decreased, implying that suppressor mutation may inactivate the quality control system. Earlier works revealed that mistranslation could provide a growth advantage in response to stress Gu et al. Hence, the decreased fidelity of translation initiation and elongation may result from the SRP deletion stress response. Increasing evidence has supported the notion that the translation elongation of nascent polypeptide regulates the targeting of SRP-dependent proteins du Plessis et al.

Decreasing the translation elongation rate extends the time window for protein targeting, which plays a critical role in suppressing the loss of SRP Zhao et al. The maximal SRP binding site is 55 amino acids from the ribosomal peptidyl transferase center in E. Thus, with the help of SRP, most translating ribosomes move to the membrane within this period in E. To get a longer time to find the membrane, the length of translating nascent chains is more likely longer than 55 amino acids. However, the nascent chain cannot exceed a specific length as aggregation would prevent protein from being targeted Siegel and Walter, ; Flanagan et al.

Proteins with fewer transmembrane domains TMDs or longer first loop lengths have a longer critical length Zhao et al. If the targeting time of some SRP-dependent proteins exceeds 10 s, these proteins would not be targeted to the inner membrane in suppressor cells.

Taken together, this model shows that SRP greatly shortened the protein targeting time by 8 s, which minimizes the cost of targeting and maintains fast growth. Overall, our data suggest that in response to the deletion of SRP, suppressor cells attenuate translation elongation to give the translating ribosomes more time to find and target to the inner membrane. Figure 5. The SRP suppressor extends the time window for protein targeting.

The suppressor cells with a slower elongation rate extended the time window for protein targeting to 2—10 s. As expected, the suppressor mutation can partially offset the defective targeting of inner membrane proteins Figure 4B , which is consistent with the previous result Zhao et al.

However, proper localization of these proteins cannot bypass the requirement of SRP Phillips and Silhavy, We speculated that the proteins that could be correctly located in the suppressor strain MY but not in SRP depletion strain SRP — may be responsible for cell survival. We hypothesized that specific membrane protein targeting defects could block the essential cellular process, which would be responsible for the loss of cell viability. Among these localization defective proteins, only one protein PgsA is essential for E.

PgsA catalyzes the step in the synthesis of the acidic phospholipids that are considered to be indispensable in multiple cellular processes Gopalakrishnan et al.

We inferred that mislocalization of PgsA inhibited cell growth. More studies are needed to investigate the targeting of some proteins that determine whether cells can survive without SRP.

The SRP-dependent delivery pathway is essential for membrane protein biogenesis. Previously, we reported that SRP was non-essential in Escherichia coli , and slowing translation speed played a critical role in membrane protein targeting.

Here, we identified a novel SRP suppressor that is also involved in translation. We found that translation speed and accuracy regulate membrane protein targeting. A slowdown of translation speed extended the time window for protein targeting. Meanwhile, a moderate decrease in translation fidelity ensured a suitable translation speed for better cell growth.

These results argued that translation control could be a practical way to compensate for the loss of SRP. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

LZ and YC performed the experiments. LZ and DZ wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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