Talkington et al.
2005
Supplementary Methods
Preparation of 16S rRNA and 30S ribosomal proteins. Native 30S subunits were purified essentially according to established methods 1-3 from MRE600 cells (American
Type Culture Collection 29417) grown to early log phase in glucose minimal media with either unlabeled or 15 N-(NH
4
)
2
SO
4
.
The 16S rRNA and the mixture of 30S ribosomal proteins were prepared by precipitating 16S rRNA from 30S subunits according to a modification of established procedures 4-6 . To a solution of 30S subunits was added an equal volume of 2x LiCl/urea
(8 M urea, 6 M LiCl, 25 mM Tris-HCl pH 7.5, 100 mM KCl, 20 mM MgCl
2
, 2 mM
DTT). The extraction reaction was incubated on ice overnight to precipitate the 16S rRNA. The RNA was pelleted by spinning at 16,000 x g at 4

C for 15 min. The pellets were washed with cold 1x LiCl/urea, held on ice for at least 1 hr, and spun for 5 min.
The mixture of ribosomal proteins was prepared by combining the supernatants from the extraction, dialyzing against TKMD (25 mM Tris-HCl pH 7.5, 1 M KCl, 20 mM
MgCl
2
, 2 mM DTT), and concentrating in YM-3 Centripreps (Millipore). Aliquots were flash frozen and stored at –80

C. The 16S rRNA was prepared by dissolving the LiCl pellets in 10 mM Tris-HCl pH 7.5 / 100 mM KCl (~0.3 mL/nmol 16S); the solution was heated at 42

C for 5 min. and held at room temperature to dissolve the RNA. Aliquots were stored as ethanol precipitations at –20

C.
The 16S rRNA was prepared for assembly by redissolving to ~3.5 mM in TKM (25 mM Tris-HCl pH 7.5, 30 mM KCl, 20 mM MgCl
2
) at room temperature and with heating at 42

C for 5 min, then cooling slowly by dialyzing against room temperature TKM at 4
1

Talkington et al.
2005

C. Proteins frozen in TKMD were thawed on ice and used directly. Concentrations of
30S, 16S, and ribosomal proteins were determined by UV absorbance (16S and 30S:

260 nm
= 12.8 x 10 6 M -1 cm -1 ; proteins:

230 nm
= 1.22 x 10 6 M -1 cm -1 ).
Pulse-chase assembly of 30S subunits . The 16S and 15 N-proteins were preheated at the temperature of the assembly reaction before mixing. The standard conditions of the assembly reaction were 0.3

M 16S and 0.45

M 15 N-proteins in assembly buffer (25 mM Tris-HCl pH 7.5 at room temperature, 330 mM KCl, 20 mM MgCl
2
, 2 mM DTT) 6 .
The binding of the 15 N-proteins was chased by adding 5x unlabeled ( 14 N) proteins. The chase was always done at 40

C for 40 min. regardless of the temperature of the pulse.
Nonspecific binding of the excess proteins in the chase was resolved by purifying the assembled 30S subunits in 10–40% sucrose gradients containing a high salt concentration
(assembly buffer with 0.5 M NH
4
Cl), analogous to the NH
4
Cl treatment of crude 70S particles used to remove bound nonribosomal proteins during preparation of the native ribosomes. The gradients were spun in an SW 41 rotor (Beckman) at 35,000 rpm at 4

C for 9 hr.
MALDI sample preparation. The subunit peaks in sucrose gradients were concentrated and buffer exchanged with assembly buffer using Centricon YM-100 concentrators
(Millipore) to reduce the sucrose concentration to < 1%. The 16S rRNA was then precipitated from 4 pmol assembled 30S subunits by adding MgCl
2
to 100 mM and 2 volumes glacial acetic acid
4
. The tubes were incubated on ice for 45 min and spun at
16,000 x g at 4

C for 15 min to pellet the rRNA, and the pellets were washed with cold
2

Talkington et al.
2005
67% acetic acid / 100 mM MgCl
2
. The 30S proteins were precipitated from the combined supernatants by adding 5 volumes acetone and incubating at –20

C for at least
2 hr. The precipitated proteins were pelleted, dried, and desalted using ZipTip
C18 s
(Millipore). (The pellets were redissolved in 8 mL 25 mM Tris pH 7.5 / 6 M urea / 2 mM
DTT, and 2 mM 0.5% TFA was added. The ZipTip resin was prepared with 50% acetonitrile / 0.1% TFA and equilibrated with 0.1% TFA, the sample was loaded, the resin was washed with 5% methanol / 0.1 % TFA, and the sample was eluted with 2 mL
70% acetonitrile / 0.1% TFA.)
The extracted and desalted proteins were spotted in the sample wells of the MALDI target plate with an equal volume of sinapinic acid (Fluka) saturated in 50% acetonitrile /
0.1% TFA. The spectra were calibrated using standard proteins in nearby spots.
MALDI analysis. Background intensity of a peak at m / z = x was approximated by determining the intensity at x in a spectrum that did not contain that peak (from samples that received mock 15 N-TP30 or mock 14 N-chase); this background was subtracted from the intensity of the peak.
The errors in the relative 15 N-protein intensities (shown as error bars on the points in the progress curves) are generally the standard deviations of relative 15 N-protein intensities in standard samples from their known
15
N-/
14
N-protein compositions. For proteins whose intensities in experimental samples are considerably different from the standard samples (apparently due to the purification in sucrose gradients with assembly buffer and NH
4
Cl), the standard deviations were estimated from other proteins with more similar intensities in the standard samples.
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Talkington et al.
2005
30S proteins monitored.
The peaks observed in the MALDI mass spectra generally agree with an earlier MALDI study of E. coli ribosomal proteins 7 , the theoretical pattern of N-terminal methionine cleavage 8, 9 (except in the case of S16, in which the methionine is apparently cleaved despite a valine in the second position), and known posttranslational modifications 10-15 . Peaks corresponding to both unacetylated and acetylated
S5 are observed (“S5u” and “S5a”); different binding kinetics are observed for the two species and thus are reported separately. Peaks corresponding to S6 with 3 or 4 Glu residues at the C-terminus are observed; the binding kinetics are similar, so the data from the two are averaged. The signal from S17 and S20 is averaged because of overlap between the peaks. At 40

C, where both proteins are almost completely bound by the first timepoint, a single progress curve and rate is reported for the two. At 15

C, some difference in the binding rates is seen, so the two 14 N peaks are quantified separately.
Because the two 15 N peaks overlap into a single peak (their theoretical masses differ by only 6 Da), the 15 N intensity for each protein is estimated as half of the height of the combined peak, since the two proteins have similar total intensities.
S2 and S7 are typically not observed in these spectra. S21 is observed but a binding rate for it cannot be reported, as the 14 N-S21 chase binds nonspecifically despite the high-salt wash.
Analysis of protein binding progress curves.
The relative
15
N-protein intensity at t = 0 was held at 0.17 in the fits, and k off
was held at a small value (1 x 10
-6
min
-1
). In cases in which the fit produced an equilibrium extent of binding greater than 1, this parameter was held at 1. In cases where the fit did not match the data well (high c
2
), the error in the experimental data points was increased to match the fit. In cases where binding appears
4

Talkington et al.
2005 to be multiphasic (see Supplementary Table S1), and thus does not fit the model well, these larger errors were used to weight the data in the fits (producing larger errors in the rates), but the smaller errors based on the standard curve are shown as error bars on the data points. The data for assembly at 15

C are a composite of two experiments, one with early timepoints and one with late timepoints,
PC/QMS and gel shift analysis of Aquifex aeolicus S15 binding to a 16S rRNA
fragment. Aquifex aeolicus S15 was overexpressed in E. coli strain BL21-RIL-(DE3)
(Stratagene) grown in glucose minimal media with unlabeled or 15 N-(NH
4
)
2
SO
4
and purified in denaturing conditions by heat-induced precipitation of endogenous E. coli proteins followed by ion exchange and reversed phase chromatography. The A4 fragment of A. aeolicus , analogous to the T4 fragment of T. thermophilus 16 , was transcribed in vitro from a plasmid using T7 RNA polymerase. The rate of S15:A4 binding (0.1

M:0.1

M) was measured using both PC/QMS and standard electrophoretic gel mobility shift techniques. Gel shift analysis was carried out according to established methods 17, 18 . S15 was incubated with 32 P-A4 (pre-incubated at 90

C for 1 min followed by ice) in 10 mM K-HEPES pH 7.5 / 50 mM KoAc / 0.1 mM EDTA / 0.1 mg/mL tRNA / 5 mg/mL heparin / 0.01% igepal at room temperature. At timepoints, an aliquot of the binding reaction was added to a 10-fold excess of unlabeled A4 and run on a nondenaturing polyacrylamide gel, and bound and free RNA bands were quantified on a
Phosphorimager. For PC/QMS of S15:A4, to avoid non-specific binding of the chase proteins, A4 RNA was added in an amount equal to the
14
N-S15 chase so that all of the chase proteins would have specific binding partners, and the A4 used in the pulse was tagged with biotin so that those complexes could be isolated. A4(biotin):S15 complexes
5

Talkington et al.
2005 were isolated on Ultralink Immobilized NeutrAvidin

resin (Pierce). The ratio of 15 N- and 14 N-S15 in the isolated complexes was analyzed by LC-MS (liquid chromatographymass spectrometry) using an HP series 1100 MSD with single ion monitoring of the four most intense charge states.
6

Talkington et al.
2005
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