Introduction
SRP recognizes ribosome emerging chain complexes (RNCs) with a transmembrane domain (TMD) or signal sequence on the NC and, via interaction with SR, delivers the RNCs to the Sec61p translocase on the ER (or SecYEG on the prokaryotic plasma membrane). Therefore, it was suggested that NAC blocks the non-specific binding of SRP or Sec61p to the ribosome.
Results
NAC also increased the specificity of SRP-SR association on RNC(ss) relative to RNC(ssmt2) from 20- to 100-fold, similar to the effects of HSW(RRL) (Fig. 1.2g vs. 2c). To quantitatively measure the effects of NAC on the binding of SRP to RNC, we performed RNC-SRP FRET titrations in the presence of increasing NAC concentrations (Fig. 1.3e,f).
Discussion
This improves the discrimination between ribosomes with and without a functional signal sequence during SRP-SR assembly, increasing the specificity of cotranslational protein targeting. The presence of NAC at the ribosome entry site does not preclude SRP binding on the same RNCs, but likely displaces SRP54-NG from its docking site near uL23 and selectively eliminates the proximal conformation for SRPs bound to RNCs with a weak signal sequence or a short nascent stage. chain.
Materials and Methods
Conversely, the predicted SRP concentration dependence of the apparent Kd,NAC values (Kd) from the anti-cooperative and competitive models were simulated using Equation (12) and Equation (13), respectively. RNCi is the RNC carrying an NC of length i, the values of [SRP]0, [SR]0, kelongation, ktarget, kon,SRP and koff,SRP are given in fig.
Supplementary Figures and Tables
The structure of the RNCss•NAC complex (Fig. 2.1A-D) reveals interactions between the N-terminal tail of NACβ and the ribosome at 3.5 Å resolution. Additionally, secretion of a ssGFP (25) was significantly lower in both mutant worms compared to wild-type NAC worms (Fig. 2.3G and Fig. S2.18C. The defects observed with SRP54mt were not for due to.Single-copy transgene Integration was performed using the miniMos103 transposon method.The RNAi-resistant gene encoding C.
The lines are fits of the data to eq 2, and the obtained Kd values are summarized in Figure 2.2H. S2.9: Additional checks related to Figure 2.3A. A) Coomassie gel showing protein samples used for analysis in Figure 2.3A. NAC cysteine variants were adjusted to the same concentration and a control gel was run under reducing conditions before performing the assay. With the ribosome, addition of NAC decreases HSP70 activity, but saturates to ~0.4× the original rate once the NAC concentration reaches the ribosome concentration (Fig. 3.1D, blue). Dual recognition of the ribosome and signal recognition particle by the SRP receptor during protein targeting to the endoplasmic reticulum.
Main Text
Charge reversal mutations of one positively charged residue in the helices (K78E-NACα or K43E- NACβ) impaired ribosome binding of NAC in vitro (Fig. S2.6A) and in vivo (Fig. The residual binding of NAC KK-EE to the ribosome is likely mediated by the N-terminus of NACβ, the position of which would not interfere with SRP binding (Fig. S2.6C). To test this, we compared the affinity of NAC for RNCs that either have an ER- signal sequence (RNCSS) displayed ) or a mutated signal sequence that inhibits ER targeting (RNCSSmt) (Fig. S2.7)10.
As predicted, adjacent cysteines can form a disulphide bond upon oxidant treatment, but only in the presence of the ribosome (Fig. 2.2A and Fig. S2.9). Cross-linking was dependent on nascent chain length and was observed only once the targeting signal emerged fully (Fig. S2.10A). The density for the NACβ anchor was still on the ribosome, in a similar position as observed in the RNC-NAC complexes (Fig. S2.12C).
Materials and Methods
Images were then subjected to 3D focused classification without adjustments by applying a circular mask to the exit site of the ribosome tunnel (pixel size of 6.048 Å, T=4, limit resolution in E-step to 25 Å), yielding RNCSS•SRP •NAC and of RNCSS • The NAC classes. To improve the resolution of the EM density corresponding to SRP-NAC and NAC, respectively, images were then subjected to an additional round of 3D focused classifications without adjustments by applying a circular mask to the SRP54 and NAC density (pixel size of 1.08) Å, T=8). 3D classification without aligning the particles into 6 classes yielded a class of active 80S ribosomes with a P-site tRNA and strong density for TTC5 and NAC (313,466 particles).
3D-classification without alignment of the counted particles into 4 classes yielded a class of active 80S ribosomes with P-site tRNA and strong density for TTC5 and NAC (83,053 particles) and this subset was refined to an angular accuracy of 0.41°. The model was then fitted to the EM-density and fitted based on the visible side chains in the cryo-EM density of the RNCSS•SRP•NAC complex. The initial framework of SRP colocalization in RNC-NAC was identified by visual inspection of fluorescence time traces.
Supplementary Figures and Tables
Light gray background indicates part of the nascent chain buried in the ribosomal tunnel (~30 aa). Therefore, to minimize disruption of the ribosome binding and structure of the RAC dimer, we decided to delete either the C-terminal domains of DNAJC2 or the NBD of HSPA14. The J domain places the HSP70-SBD right on top of the ribosomal tunnel exit and is close to the tip of the ZHD.
Regulation by the ribosome of the GTPase of the signal recognition particle during protein targeting. Dual binding mode of the nascent polypeptide-associated complex reveals a novel universal adapter site on the ribosome. Analysis of the interaction of protein biogenesis factors at the ribosome exit site reveals a new role for NAC.
Introduction
In bacteria, trigger factor (TF) is a major co-translational chaperone that forms a hydrophobic cradle just outside the ribosomal tunnel and binds to the nascent strand to prevent misfolding 106 . NAC is stoichiometrically close to the ribosome and binds to the ribosome with low nanomolar affinity, suggesting that all translating ribosomes in the cytosol are bound by NAC82. RAC binds to the ribosome primarily through DNAJC2, which spans the small (SSU) and large subunit (LSU) of the ribosome 116–118.
Mammalian RAC (mRAC) is homologous to yeast RAC and can rescue the deletion of yeast RAC when expressed through a plasmid119. The ZHD is connected to the J domain of Zuo1, placing it close to the ribosomal tunnel exit. The Zuo1 N-terminal domain is attached to the J domain through a flexible linker and directly interacts with Ssz1 to dimerize120.
Results
Deletion of the C-terminal domain of DNAJC2 up to residue 346 did not significantly perturb HSP70 activation by RAC with or without the ribosome (Fig. 3.1C, d549, d449 and d346). Interestingly, the interaction we measured using this FRET design may be the intrinsic binding between HSP70 and NC since the affinity was not significantly altered by the presence of RAC or the nucleotide (Fig. 3.2D). Importantly, this effect is specific to the presence of the active J domain, as the use of 5 μM mutant RAC QPD did not significantly alter the affinity compared to no RAC (Fig. S3.3B).
Kd from titrations like the one in Figure C) Equilibrium FRET titration between HSP70 and different RNCs. Addition of HSP70 alone in the presence of ATP did not change the kinetics or the final chemiluminescence, confirming no interaction with the NC from the ribosome (Figure 3.3B, blue). Importantly, this effect is specifically due to RAC activation of HSP70 activity, as QPD application of mutant RAC to the J domain completely abolishes the delay (Figure 3.3B, green).
Discussion and Future Directions
The absence of the C-terminal 35 aa completely inhibits NLuc activity, probably due to the inability to fold into a complete structure129. Since even ribosome-bound RAC has a considerable degree of flexibility, solving the free structure of RAC can prove very difficult. From a thermodynamic equilibrium perspective, since ribosome binding to RAC stimulates the recruitment of HSP70 to RAC in the ATPase assay, we would expect that HSP70 binding to RAC also stimulates RAC binding to the ribosome.
We observed no significant contribution of the C-terminal expansion to ribosome detection, nor to the overall cochaperon activity of RAC (Fig. 3.1C). Indeed, the surprising fact that the C-terminus and NC of DNAJC2 are in the FRET radius suggests a kind of dynamic interaction that brings the C-terminal extension close to the exit of the ribosome tunnel (Fig. S3.2). To guide NC, RAC recruits HSP70 to the ribosome to interact directly with the NC.
Materials and Methods
The NC selectivity of such HSP70 recruitment may be related to the NC affinity for HSP70 or negatively regulated by the binding of other RPBs such as SRP. The extent of hydrolysis is determined from the ratio of radioactivity of ADP to total radioactivity. The rate of hydrolysis is determined by fitting a pseudo-first-order kinetics to the data.
The ybbr labeled RAC was purified in the same manner as WT and labeled with Atto647n using Sfp labeling. RAC and HSC70 were added to the ribosome resuspension at the indicated concentrations and transferred to a 96-well plate at room temperature. Quartz slides with PEGylation and biotin modification were prepared in the same way as in Chapter 1 and 2.
Supplementary Figures and Tables
Signal recognition particle receptor mediates GTP-dependent translocation of SRP from the nascent polypeptide signal sequence. Reconstitution of the human SRP system and quantitative and systematic analysis of its ribosome interactions. The crystal structure of the human polypeptide-related complex domain reveals a nucleic acid binding region in the NACA subunit.
Conformational changes in the GTPase modules of the signal-receiving particle and its receptor trigger the initiation of protein translocation. Each of the signal recognition particle (SRP) activities is contained in a separate area: Analysis of biochemical mutants of SRP. Structure and evolution of the 4-helix cluster domain of Zuotin, a J-domain protein co-chaperone of Hsp70.
