Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape

Abstract

The Escherichia coli 30S ribosomal subunit self-assembles in vitro in a hierarchical manner, with the RNA binding by proteins enabled by the prior binding of others under equilibrium conditions. Early 16S rRNA binding proteins also bind faster than late-binding proteins, but the specific causes for the slow binding of late proteins remain unclear. Previously, a pulse-chase monitored by quantitative mass spectrometry method was developed for monitoring 30S subunit assembly kinetics, and here a modified experimental scheme was used to probe kinetic cooperativity by including a step where subsets of ribosomal proteins bind and initiate assembly prior to the pulse-chase kinetics. In this work, 30S ribosomal subunit kinetic reconstitution experiments revealed that thermodynamic dependency does not always correlate with kinetic cooperativity. Some folding transitions that cause subsequent protein binding to be more energetically favorable do not result in faster protein binding. Although 3(') domain primary protein S7 is required for RNA binding by both proteins S9 and S19, prior binding of S7 accelerates the binding of S9, but not S19, indicating there is an additional mechanistic step required for S19 to bind. Such data on kinetic cooperativity and the presence of multiphasic assembly kinetics reveal complexity in the assembly landscape that was previously hidden.

Fig. 1.

(A) Nomura equilibrium assembly map (6, 23). Protein binding dependencies at equilibrium are shown as arrows. Colored circles correspond to protein-binding rates at 15 °C as measured by PC/QMS: 7.8 - 3.1 min^-1 (Red), 0.7 min^-1 (Orange), 0.042 - 0.015 min^-1 (Green), 0.01 - 0.005 min^-1 (Blue), and 0.003 - 0.0008 min^-1 (Purple). The rates of S2 and S21 are not shown because those proteins bind transiently and hence kinetics cannot be observed using pulse-chase (16). (B) Kinetic cycle with three proteins binding an RNA, showing two potential intermediates. (C) Prebinding PC/QMS experimental scheme. Prebinding proteins are incubated with 16S rRNA at 40 °C before the pulse-chase.

Fig. 2.

(A) Kinetic assembly map from primary protein prebinding experiment. Observed kinetic cooperativity is indicated with colored arrows and colored proteins. The thickness of the arrow relates to the magnitude of the cooperativity, with large accelerations indicated with thick arrows and modest accelerations indicated with thinner arrows. Small gray arrows are from the Nomura map. (B) Protein binding progress curves for protein S6 from experiments prebinding only the primary proteins (Red) and a control experiment (Blue). All curves are single exponential fits except those marked with an asterisk, which were best fit with a double exponential. (C) Kinetic assembly map from primary and secondary protein prebinding experiment. (D) As for (B), except showing S10 progress curves and including data from an experiment prebinding both the primary and secondary proteins (Purple). (E) Kinetic assembly map from secondary proteins prebinding experiment. No kinetic cooperativity was observed, and the binding of protein S4 was inhibited (Fig. S1).

Fig. 3.

(A), Protein binding progress curves for S3 from a control experiment (Blue), the prebinding of the 5^′ and central domains (Red), and the prebinding of the 5^′ and central domains plus S7 (Green). All curves are single exponential fits except those marked with an asterisk, which are double exponential. (B) Kinetic assembly map showing no significant kinetic cooperativity with the 5^′ and central domains prebound. (C)–(D) As in (A) except showing progress curves for (C) S10 and (D) S9. (E). Kinetic assembly map for prebinding of 5^′ and central domain proteins plus S7 (Fig. S2).

Fig. 4.

(A)–(B) Protein binding progress curves for proteins (A) S9 and (B) S10 from a control experiment (Blue), prebinding of S7 (Green), prebinding of S7 and S19 (Red), and prebinding of S7 and S13 (Purple). Double exponential curves are indicated with an asterisk. (C)–(F) Kinetic assembly maps of the 3^′ domain only from experiments prebinding (C) S7 alone, (D) S7 and S13, (E) S7 and S9, (F) S7 and S19 (Fig. S3).