Temperature-dependent RNP conformational rearrangements: analysis of binary complexes of primary binding proteins with 16 S rRNA

Abstract

Ribonucleoprotein particles (RNPs) are important components of all living systems, and the assembly of these particles is an intricate, often multistep, process. The 30 S ribosomal subunit is composed of one large RNA (16 S rRNA) and 21 ribosomal proteins (r-proteins). In vitro studies have revealed that assembly of the 30 S subunit is a temperature-dependent process involving sequential binding of r-proteins and conformational changes of 16 S rRNA. Additionally, a temperature-dependent conformational rearrangement was reported for a complex of primary r-protein S4 and 16 S rRNA. Given these observations, a systematic study of the temperature-dependence of 16 S rRNA architecture in individual complexes with the other five primary binding proteins (S7, S8, S15, S17, and S20) was performed. While all primary binding r-proteins bind 16 S rRNA at low temperature, not all r-proteins/16 S rRNA complexes undergo temperature-dependent conformational rearrangements. Some RNPs achieve the same conformation regardless of temperature, others show minor adjustments in 16 S rRNA conformation upon heating and, finally, others undergo significant temperature-dependent changes. Some of the architectures achieved in these rearrangements are consistent with subsequent downstream assembly events such as assembly of the secondary and tertiary binding r-proteins. The differential interaction of 16 S rRNA with r-proteins illustrates a means for controlling the sequential assembly pathway for complex RNPs and may offer insights into aspects of RNP assembly in general.

Figure 1

1. Modified in vitro 30S subunit assembly map. The 16S rRNA is represented by a rectangle in a 5' to 3' direction. The arrows indicate the co-dependencies for the assembly of the r-proteins. The size of the arrow indicates the relative strength of the assembly dependency between components. The r-proteins shown in the white region are primary binding r-proteins. The r-proteins shown in white in the light gray and dark gray box indicate secondary, and tertiary binding r-proteins, respectively. S6 and S18 are enclosed in a box to indicate that they bind as a heterodimer.

2. Crystal structure of the 16S rRNA from the E. coli 30S subunit with all the primary proteins. The 16S rRNA is shown in gray, and the r-proteins are S4 green, S7 red, S8 magenta, S15 bright yellow, S17 dark purple and S20 light blue, as in Figure 1a. The 3-D parts of the 30S subunit are indicated while the corresponding domains from the 16S rRNA secondary structure are specified in paranthesis. All the Figures containing 3-D structures were prepared using Pymol, and the pdb file 2AW7.

Figure 2

Primer extension analysis of the r-protein/16S rRNA complexes. Individual gels of the minimal complexes modified by DMS or kethoxal are shown. A and G (lanes 1 and 2) are dideoxy sequencing lanes, K (lane 3): unmodified 16 S rRNA. All the other lanes are treated with the probe indicated below. The other lanes 4-8 are: modified 16S rRNA kept at 0°C (lane 4), or at 42°C (lane 5), Sx/16S rRNA formed at 0°C (lane 6), or at 42°C (lane 7) and the shifted complex (lane 8). Compare lanes 4 and 6 for the complexes formed at 0°C, lanes 5 and 7 for the complexes formed at 42°C, or lanes 6 and 7 for the differences between the two complexes. The probes and primers used for the experiments are indicated below. S20/16S rRNA: a) DMS-323, b) kethoxal-323, c) DMS-480, d) DMS-1508; S17/16S rRNA: e) DMS-323; f) kethoxal, 323; S15/16S rRNA: g) DMS-795, h) kethoxal-795 S8/16S rRNA: i) DMS-683, j) kethoxal-683, k) DMS-795, l) DMS-939, m) kethoxal-939, n) DMS–939; S7/16S rRNA: o) DMS-1046, p) kethoxal-1046, q) DMS-1391, r) DMS -1491, s) kethoxal -1491. The symbols x and Δ indicate temperature-independent and temperature-dependent footprints, respectively.

Figure 2

Primer extension analysis of the r-protein/16S rRNA complexes. Individual gels of the minimal complexes modified by DMS or kethoxal are shown. A and G (lanes 1 and 2) are dideoxy sequencing lanes, K (lane 3): unmodified 16 S rRNA. All the other lanes are treated with the probe indicated below. The other lanes 4-8 are: modified 16S rRNA kept at 0°C (lane 4), or at 42°C (lane 5), Sx/16S rRNA formed at 0°C (lane 6), or at 42°C (lane 7) and the shifted complex (lane 8). Compare lanes 4 and 6 for the complexes formed at 0°C, lanes 5 and 7 for the complexes formed at 42°C, or lanes 6 and 7 for the differences between the two complexes. The probes and primers used for the experiments are indicated below. S20/16S rRNA: a) DMS-323, b) kethoxal-323, c) DMS-480, d) DMS-1508; S17/16S rRNA: e) DMS-323; f) kethoxal, 323; S15/16S rRNA: g) DMS-795, h) kethoxal-795 S8/16S rRNA: i) DMS-683, j) kethoxal-683, k) DMS-795, l) DMS-939, m) kethoxal-939, n) DMS–939; S7/16S rRNA: o) DMS-1046, p) kethoxal-1046, q) DMS-1391, r) DMS -1491, s) kethoxal -1491. The symbols x and Δ indicate temperature-independent and temperature-dependent footprints, respectively.

Figure 2

Primer extension analysis of the r-protein/16S rRNA complexes. Individual gels of the minimal complexes modified by DMS or kethoxal are shown. A and G (lanes 1 and 2) are dideoxy sequencing lanes, K (lane 3): unmodified 16 S rRNA. All the other lanes are treated with the probe indicated below. The other lanes 4-8 are: modified 16S rRNA kept at 0°C (lane 4), or at 42°C (lane 5), Sx/16S rRNA formed at 0°C (lane 6), or at 42°C (lane 7) and the shifted complex (lane 8). Compare lanes 4 and 6 for the complexes formed at 0°C, lanes 5 and 7 for the complexes formed at 42°C, or lanes 6 and 7 for the differences between the two complexes. The probes and primers used for the experiments are indicated below. S20/16S rRNA: a) DMS-323, b) kethoxal-323, c) DMS-480, d) DMS-1508; S17/16S rRNA: e) DMS-323; f) kethoxal, 323; S15/16S rRNA: g) DMS-795, h) kethoxal-795 S8/16S rRNA: i) DMS-683, j) kethoxal-683, k) DMS-795, l) DMS-939, m) kethoxal-939, n) DMS–939; S7/16S rRNA: o) DMS-1046, p) kethoxal-1046, q) DMS-1391, r) DMS -1491, s) kethoxal -1491. The symbols x and Δ indicate temperature-independent and temperature-dependent footprints, respectively.

Figure 2

Primer extension analysis of the r-protein/16S rRNA complexes. Individual gels of the minimal complexes modified by DMS or kethoxal are shown. A and G (lanes 1 and 2) are dideoxy sequencing lanes, K (lane 3): unmodified 16 S rRNA. All the other lanes are treated with the probe indicated below. The other lanes 4-8 are: modified 16S rRNA kept at 0°C (lane 4), or at 42°C (lane 5), Sx/16S rRNA formed at 0°C (lane 6), or at 42°C (lane 7) and the shifted complex (lane 8). Compare lanes 4 and 6 for the complexes formed at 0°C, lanes 5 and 7 for the complexes formed at 42°C, or lanes 6 and 7 for the differences between the two complexes. The probes and primers used for the experiments are indicated below. S20/16S rRNA: a) DMS-323, b) kethoxal-323, c) DMS-480, d) DMS-1508; S17/16S rRNA: e) DMS-323; f) kethoxal, 323; S15/16S rRNA: g) DMS-795, h) kethoxal-795 S8/16S rRNA: i) DMS-683, j) kethoxal-683, k) DMS-795, l) DMS-939, m) kethoxal-939, n) DMS–939; S7/16S rRNA: o) DMS-1046, p) kethoxal-1046, q) DMS-1391, r) DMS -1491, s) kethoxal -1491. The symbols x and Δ indicate temperature-independent and temperature-dependent footprints, respectively.

Figure 3

Nucleotides with altered reactivity as a result of binding of an r-protein to 16S rRNA, for both kethoxal and DMS probing represented on the secondary structure of 16S rRNA. Circles denote the sites of protections, while squares denote enhancement sites, and the size represents the intensity of the change. The 16S rRNA is shown in dark gray, changes attributed to: S4, green; S7, red; S8, magenta; S15 bright yellow; S17, dark purple, and S20, light blue. Nucleotides enhanced or protected by more than one protein are shown as concentric rings or squares. (a) Changes in modification patterns shown on the secondary structure of 16 S rRNA for complexes formed at 0°C. b) Changes in modification patterns shown on the secondary structure of 16 S rRNA for the interaction at 42°C. c) Difference in the nucleotides with altered reactivity between the complexes formed at 42°C and 0°C.

Figure 4

Details of footprints for r-proteins S15 and S8. The 16S rRNA is shown in gray, S15 and S8 are shown in rainbow, from blue (N-terminus) to red (C-terminus). Footprints which are not temperature-dependent are shown in blue, footprints that appear at 0°C and continue to develop in intensity at 42°C are shown in purple, and footprints that appear only at 42°C are shown in red. a) 16S rRNA r-protein and S15 from the crystal structure of E. coli 30S subunit, b) S15-dependent protections; c) S15-dependent enhancements; d) 16S rRNA r-protein and S8 from the crystal structure of E. coli 30S subunit, e)S8-dependent protections; f) S8-dependent enhancements.

Figure 5

Details of footprints for r-proteins S4 and S7. The 16 S rRNA is shown in gray, S15 and S8 are shown in rainbow, from blue (N-terminus) to red (C-terminus). Coloring of footprints as described in Figure 4. a) 16S rRNA r-protein and S4 from the crystal structure of E. coli 30S subunit, b) S4-dependent protections; c) S4-dependent enhancements; d) 16S rRNA r-protein and S7 from the crystal structure of E. coli 30S subunit, e) S7-dependent protections; f) S7-dependent enhancements.

Figure 6

The temperature-dependent footprints for each primary r-protein represented on the crystal structure of 16S rRNA from the E. coli 30S subunit. Nucleotides with altered reactivities are represented as spheres. The size of the spheres is indicative of the intensity of the change. The r-proteins are omitted for clarity.