Signal recognition particle-ribosome binding is sensitive to nascent chain length

Abstract

The signal recognition particle (SRP) directs ribosome-nascent chain complexes (RNCs) displaying signal sequences to protein translocation channels in the plasma membrane of prokaryotes and endoplasmic reticulum of eukaryotes. It was initially proposed that SRP binds the signal sequence when it emerges from an RNC and that successful binding becomes impaired as translation extends the nascent chain, moving the signal sequence away from SRP on the ribosomal surface. Later studies drew this simple model into question, proposing that SRP binding is unaffected by nascent chain length. Here, we reinvestigate this issue using two novel and independent fluorescence resonance energy transfer assays. We show that the arrival and dissociation rates of SRP binding to RNCs vary according to nascent chain length, resulting in the highest affinity shortly after a functional signal sequence emerges from the ribosome. Moreover, we show that SRP binds RNCs in multiple and interconverting conformations, and that conversely, RNCs exist in two conformations distinguished by SRP interaction kinetics.

Keywords: Fluorescence Resonance Energy Transfer (FRET); Protein Targeting; Ribosome Function; Signal Recognition Particle (SRP); Single-Molecule Biophysics.

FIGURE 1.

Protein-labeled FRET assay to detect SRP-RNC interactions. A, L29 ribosome reconstitution scheme. B, L29 ribosomes were separated into 30S and 50S subunits in a 1 mm magnesium acetate 10–40% sucrose gradient. Top panel shows A_{260 nm} of collected gradient fractions. Bottom panel shows fluorescence visualization of Cy3-labeled L29 in fractions analyzed with denaturing PAGE. C, incorporation of Cy3-labeled L29 into ΔL29 but not WT ribosomes as determined after ribosomes were incubated with L29 and pelleted through a 40% sucrose cushion of L29 wash buffer. The pelleted ribosomes were assayed for absorbance at 260 and 550 nm (average of four separate L29 ribosome preps, error bars indicate 1 S.D.). D, translation products of WT or L29 ribosomes using truncated mRNA for 35-aa and 75-aa lepB mRNA after a 2-h translation in PURE system. Stalled RNCs were isolated after spinning over a 40% sucrose 400 mm NH₄Cl cushion. Half of the stalled RNCs were visualized via autoradiography of [³⁵S]Met incorporation after denaturing PAGE. The other half of the stalled RNCs were quantified using a scintillation counter to compare translation efficiency. E, SR-dependent GTPase activity of different Cy-labeled SRP constructs as compared with unlabeled WT SRP. SRP GTPase activities derived from fits are as follows: 0.99 ± 0.07 s⁻¹ (± S.E.) for WT, 0.51 ± 0.06 s⁻¹ for protein-labeled SRP, 1.05 ± 0.03 s⁻¹ for nucleotide-labeled SRP.

FIGURE 2.

Ensemble equilibrium measurements reveal that SRP-RNC binding is sensitive to nascent chain length. All results shown use the protein-labeled assay. A, K_d measurements of SRP-RNC binding when the RNC was stalled with different aa length lepB nascent chains. Cy5-labeled SRP was incubated with Cy3B-labeled RNCs. Each point is the average of triplicate experiments with error bars indicating S.D. Data were fitted to quadratic binding curves with saturation E_FRET set at 0.17, yielding K_d values of 1.38 ± 0.23 nm (± S.E.) for 35-aa RNC, 0.132 ± 0.012 nm for 55 aa, 0.057 ± 0.010 nm for 75 aa, 0.060 ± 0.013 nm for 95 aa, 0.163 ± 0.022 nm for 105-aa RNC, and 0.299 ± 0.057 nm for 135 aa. B, summary of K_d values determined in A. Note that the y axis is split to accommodate values >0.35 nm.

FIGURE 3.

SRP arrives to its RNC in distinct and interconverting conformations. All results shown use the protein-labeled assay, panels A and B used TIRFM, and panels (C–F) used ZMWs. A, example single-molecule trace of Cy5-labeled SRP delivered at time = 0 to slides with immobilized Cy3-labeled 75-aa RNC. The top panel shows the fluorescence intensity of the Cy3 (green) and Cy5 (red) signal. The bottom panel shows the FRET efficiency of the two signals. Dots indicate FRET-binding events, and the line indicates a Cy3-blinking event. The trace shown presented more than the ∼2 event/trace average. AU indicates arbitrary units. B, histogram of the average E_FRET values of binding events observed in traces similar to the one shown in A. Lines indicate the normal fits of low E_FRET events (red), intermediate E_FRET events (green), and high E_FRET events (blue). The percentage of total events and mean (± one S.D.) E_FRET value for each are indicated (n = 465). C and D, single-molecule traces of Cy5-labeled SRP delivered at time = 0 to slides with immobilized Cy3-labeled 75-aa RNC. Triangles show transitions between different FRET states. E, histogram of the average E_FRET values of the distinct FRET states assigned in SRP-RNC binding events with FRET transitions. Lines indicate the normal fits of the low E_FRET events (red) intermediate E_FRET events (green) and high E_FRET events (blue). The percentage of total events and mean E_FRET value (± one S.D.) for each type of binding are indicated (n = 218. 42 binding events showed transitions, each of those events had ∼3 transitions yielding ∼5 distinct states per binding event). F, E_FRET transition density plot showing the average E_FRET of the distinct FRET states from the experiment described in C–E. INT., intermediate E_FRET events.

FIGURE 4.

Nucleotide-labeled smFRET assay to detect SRP-RNC interactions. All results shown use the nucleotide-labeled assay. A, single-molecule trace of Cy5-labeled SRP delivered at time = 0 to slides with immobilized Cy3B-labeled 75-aa RNC visualized using TIRFM. The fluorescence intensity of the Cy3B (green) and Cy5 (red) signals is shown. FRET events, arrival time, and residence times are labeled. AU, arbitrary units. B, histogram plot with normal fit of observed E_FRET values when SRP was delivered to 75-aa RNC. For the experiment shown in this panel, the 50S subunits were unlabeled (see “Experimental Procedures”). C, bar plot of observed SRP-RNC FRET events and their dependence on aa length of nascent chains on stalled RNC, nature of the signal sequence (WT or mutant (MUT) (see “Experimental Procedures” for sequence)), and composition of SRP (WT or 4.5S RNA alone (RNA). Ø indicates that no events were measured. D, cumulative distribution of lifetimes of the dye on SRP when SRP was directly immobilized on the slide surface. E and F, cumulative distributions of arrival times (E) and residence times (F) of SRP binding to 75-aa RNC. Gray and red lines indicate fits to single and double exponentials, respectively. The percentage and average (AVG.; ± 95% confidence interval of fit) arrival and residence times to the double exponential for each type of event are indicated (n ≥ 419 binding events).

FIGURE 5.

smFRET kinetic measurements confirm that SRP-RNC binding is sensitive to nascent chain length. All results shown use the nucleotide-labeled assay. A and B, cumulative distributions of arrival times (A) and residence times (B) of SRP binding events to RNCs with different length nascent chains. Insets show a magnification of the curves within the dashed lines. Experiments were performed as described (for Fig. 4A). Dashed lines indicate fits to double exponential distributions (n ≥ 192). C, K_d(app) values for SRP-RNC binding to RNC stalled with different length nascent chains derived from the data in (A and B). Each colored line connects affinities determined by the same pairing of binding rates: fast arrivals with either long (blue), or short (red) residence times and slow arrivals with either long (green), or short (purple) residence times. Error bars indicate error propagated from the ± 95% confidence interval of fits for the data shown in A and B from which the affinities were estimated. D, fold difference of affinities shown in C. Colors are the same as in C. Note that the y axis is split to accommodate fold differences larger than 9.