THE JOURNAL OF BIOLOGICAL CHEMISTRY Val. 258, No. 10, Issue of May 25, pp. 6313-6318, 1983 Printed in U . S . A .

Reverse Phase High Performance Liquid Chromatography of Escherichia coli Ribosomal Proteins: Standardization of 70 S, 50 S, and 30 S Protein Chromatograms FUNCTIONAL ACTIVITY OF PURIFIED PROTEINS*

(Received for publication, December 7, 1982)

Anthony R. KerlavageS, Tayyaba Hasan§, and Barry S. Coopermanll From the ‘Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104

We recently described the use of reverse phase high performance liquid chromatography for the separation of the proteins of the 30 S subunit of Escherichia coli ribosomes (Kerlavage, A. R., Kahan, L., and Cooper- man, B. s. (1982) Anal. Biochem. 123, 342-348). In the present studies we report improvements in the technique and its extension to the separation of the proteins of the 50 S subunit and of 70 S ribosomes. Using an octadecasilyl silica column and a trifluoro- acetic acidlacetonitrile solvent system, the 21 proteins of the 30 S subunit have been resolved into 17 peaks, the 33 proteins of the 50 S subunit into 22 peaks, and the 53 proteins of the 70 S ribosome into 31 peaks. The proteins present in each peak have been identified by polyacrylamide gel electrophoresis, by comparison with previously standardized chromatograms, and by calibration with authentic samples of purified proteins. All of the known ribosomal proteins have been identi- fied on the chromatograms with the exception of L31 and its variant, L31’. Three protein peaks, not corre- sponding to known ribosomal proteins, have been ob- served in preparations from the total protein from 50 S subunits and 70 S ribosomes, but the significance of these peaks is unclear.

The reverse phase high performance liquid chroma- tography technique has the potential for purifying all ribosomal proteins, as demonstrated by the increase in resolution we obtain when a peak isolated under stand- ard gradient conditions and containing several pro- teins is reapplied to the column and eluted with a shallower gradient. Its utility in preparing proteins for functional studies is demonstrated by a reconstitution of active 30 S particles using 30 S proteins prepared by reverse phase high performance liquid chromatog- raphy.

The Escherichia coli ribosome has a sedimentation coeffi- cient of 70 S and consists of two dissociable subunits with sedimentation coefficients of 30 S and 50 S. The 30 S subunit is composed of 16 S RNA and 21 proteins, while the 50 S subunit is composed of 23 S RNA, 5 S RNA, and 33 proteins.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertzsernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of United States Public Health Service Postdoctoral Fellowship GM-08966.

Q Present address, Photomedicine Research Unit, Department of Dermatology, Massachusetts General Hospital, Boston, MA 02114.

7 Supported in part by United States Public Health Service Grant AI-16806.

The proteins derived from the small (30 S) subunit are des- ignated S1-S21 and those derived from the large (50 S) subunit are designated Ll-L34. There are a total of 53 unique proteins; L8 is a stable complex of L7/L12 and L10 (1) and S20 and L26 are identical (a), one copy of this protein being partitioned between the 30 S and 50 S subunits (3).

The preparative and analytical separation of ribosomal proteins is of crucial importance to the study of the structure and function of the ribosome. Various approaches requiring purification or analysis of ribosomal proteins have been em- ployed in these studies. These include reconstitution of ribo- somal subunits from constituent components with the omis- sion of a single protein (4, 5), immunoelectron microscopy using antibodies to individual proteins (6, 71, affinity labeling of the ribosome with derivatives of its various ligands (8-lo), neutron diffraction using selectively deuterated proteins (1 l ) , assembly mapping (reviewed in Ref. 12), protein cross-linking (13), and analysis of mutants with altered or missing proteins (reviewed in Ref. 14).

The major problem in separating the ribosomal proteins is that most of them are of similar molecular mass (6,000 to 30,000 Da) and isoelectric point (8 to 12) (15). The classical purification methods for ribosomal proteins include ion ex- change and size exclusion chromatography steps. These pro- cedures are quite laborious and yields range from 5% for the smaller, difficult to isolate proteins to 50% for the larger proteins (16-19). The classical analytical methods for the separation of ribosomal proteins have been one- and two- dimensional polyacrylamide gel electrophoresis. One-dimen- sional electrohoresis performed according to Leboy et al. (20) resolves the 21 proteins of the 30 S subunit into 15 bands and the 33 proteins of the 50 S subunit into 16 bands. For the case of analysis of radioactively labeled proteins, the time required for pouring, running, staining, destaining, and drying the gel and then oxidizing gel slices prior to scintillation counting is quite substantial. In addition, the recovery of covalently incorporated radioactivity is usually below 50% (21). Two-dimensional polyacrylamide gel electrophoresis, performed as introduced by Kaltschmidt and Wittmann (22) or using more recent modifications (23, 24), resolves almost all of the 30 S and 50 S proteins but with an even greater time expenditure and lower recovery of protein.

Reverse phase high performance liquid chromatography is well suited to separating the ribosomal proteins since the basis for retention is hydrophobicity rather than size or charge. RP-HPLC’ also has the advantages of speed, high

The abbreviations used are: RP-HPLC, reverse phase high per- formance liquid chromatography; TP30, TP50 and TP70, total pro- tein from 30 S and 50 S subunits and 70 S ribosomes, respectively; PAGE, polyacrylamide gel electrophoresis.

6313

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63 14 RP-HPLC of E. coli Ribosomal Proteins

recovery yields, and high sensitivity. We previously reported on the separation of TP30 by RP-HPLC into a total of 15 peaks, and on the identification of the 30 S proteins within each of these peaks (25). In the present work we report several technical improvements in the RP-HPLC technique, use our improved methodology to separate TP50, TP30, and TP70 into large numbers of peaks, and identify the proteins eluting in each of the separated peaks. Our results clearly demonstrate the superiority of this technique to classical methods for both preparative and analytical separation of ribosomal proteins.

EXPERIMENTAL PROCEDURES

Materials-Trifluoroacetic acid and HPLC grade acetonitrile were purchased from Fisher. Lyophilization jars were siliconized with Prosil-28 (PCR Chemicals). Poly(U) was purchased from Miles. ["C] Phe.tRNAPhe was prepared from [14C]Phe (400-450 Ci/mol, New England Nuclear) and bulk-stripped tRNA (Grand Island Biological) as described by Ravel and Shorey (26). Coomassie brilliant blue G- 250 and bovine serum albumin were purchased from Sigma. All other chemicals were reagent grade.

Purified E. coli 50 S ribosomal proteins were a generous gift of Dr. M. Nomura, University of Wisconsin. Ribosomal proteins L7/L12 were a generous gift of Dr. A. Dahlberg, Brown University.

Isolation of Ribosomal Proteins-70 S ribosomes were prepared from E. coli Q13 bacteria harvested in mid or late log phase using the modification of the Traub et al. (27) procedure previously described (21). Ribosomal subunits were prepared by sucrose gradient centrif- ugation as described previously (21) using buffer A (50 mM Tris-HC1 (pH 7.6 at 4 "C), 50 mM KCl, 1 mM MgClZ, 6 mM 2-mercaptoethanol). Protein was extracted from 70 S, 50 S, and 30 S particles using the Me-acetic acid procedure of Hardy et al. (16) and was precipitated with acetone (28). Precipitates were dissolved in buffer B (6 M urea, 150 mM LiCl, 10 mM (adjusted to pH 8.0 with methylamine), 3 mM 2-mercaptoethanol) prior to HPLC injection.

High Performance Liquid Chromatography-The HPLC system consisted of one 6000 A pump, one "45 pump, a 660 programmer, and a U6K universal injector, all from Waters Associates. Column eluates were monitored for UV absorbance using a Waters extended wavelength module (214 nm) and a model 440 absorbance detector (280 nm) connected in series. Each was equipped with a 15.5-pl cell with a 1-cm path length. All separations were performed on a Syn- chropak RP-P Cls-silica column (6.5 pm silica, 300-A pore, 4.1 X 250 mm; SynChrom, Inc.).

Proteins were eluted at room temperature using a gradient (as described in Figs. 1, 4, and 6) from 0.1% (w/v) F3CCOOH in H20, pH 2.14 (solvent A) to 0.1% (w/v) F3CCOOH in CHSCN (solvent B). The "45 was used to pump solvent A and the 6000 A was used to pump solvent B at a combined constant flow rate of 0.7 ml/min with column pressure between 500 and 1500 p.s.i.

Solvent A was prepared from deionized, reversed osmosis-purified HzO which was filtered twice under aspirator vacuum through 0.45- pm Metricel filters (Gelman Sciences) placed in a 0.5-pm sintered glass Millipore vacuum filter. FaCCOOH was added after filtration. CH&N was filtered once through 0.2-pm nylon 66 filters (Rainin Instruments) prior to addition of F3CCOOH. Solvents were stirred constantly during runs and degassed approximately every 24 h by filtering as above. Columns were returned to initial conditions by a 10-min linear gradient a t a flow rate of 2 ml/min and equilibrated for an additional 10 min at 2 ml/min.

Little change in the chromatographic pattern obtained was ob- served on variation of the total protein applied in a single run between 10 pg and 2 mg. Minor losses in resolution were observed on appli- cation of up to 5 mg of protein. The overall yield of protein recovered from a typical HPLC run was 85%, in agreement with our previous result (25). Protein determinations were made according to Bradford (29) using bovine serum albumin as a standard.

Polyacrylamide Gel Electrophoresis-One-dimensional urea-poly- acrylamide gels were run according to Leboy et al. (20). Two-dimen- sional urea-polyacrylamide gels were run according to Kenny et al. (24). Assignment of 50 S and 30 S protein migrations within the gels followed those of Mora et al. (30) and Rummel and Noller (31), respectively. Fractions from HPLC runs were lyophilized and redis- solved in running buffer (8 M urea, 10 mM 2-mercaptoethanol) before application to the gels.

30 S Reconstitution-Non-HPLC- or HPLC-treated TP30 (2.8 nmol) was reconstituted with 16 S RNA (2.3 nmol) as described by

Traub et al. (27). TP30 was prepared either by MP-acetic acid extraction, with the acetone precipitate redissolved in 8 M urea, or by LiC1-urea extraction (17). In either case, the urea suspension of protein was exhaustively dialyzed against buffer C (5 mM H3PO4 (adjusted to pH 7.5 with KOH at 4 "C), 20 mM MgC12, 1 M KCl, 6 mM 2-mercaptoethanol) prior to reconstitution with 16 S RNA. HPLC-treated protein was prepared by applying an aliquot of TP30 in buffer C to the Cls-silica column and eluting the protein as described above. The total eluate was collected in a siliconized jar and lyophilized. The lyophilized protein was taken up in 4 M urea and 2 M LiCl and dialyzed against buffer C prior to reconstitution with 16 S RNA.

tRNA Binding-Phe.tRNAPhe binding to 30 S subunits was as- sayed as described previously (32). Concentrations in the binding assay were: 30 S subunits, 0.78 pM; [l4C]Phe.tRNAPhe, 0.15 p ~ ; poly(U), 0.24 pglml.

Sucrose Density Gradient Centrifugation-Reconstituted and na- tive 30 S subunits (80-200 pmol) were centrifuged through linear 15- 30% sucrose gradients made up in buffer A in a Beckman VTi50 rotor at 4 "C as described elsewhere (33).

RESULTS AND DISCUSSION

Since our previous work on the separation of TP30 by RP- HPLC (25), four important technical improvements have been made: (a) utilization of a 300-A pore Cle-silica column (Synchropak RP-P, SynChrom, Inc.) replacing the 125-A pore column (p-Bondpak, Waters Associates) used earlier; (b) changing the gradient shape from linear to convex; (c) addi- tion of 0.1% F,CCOOH to the acetonitrile solvent; ( d ) dual wavelength monitoring a t 214 and 280 nm. The results of applying our improved procedure to separations of TP50, TP30, and TP70 are discussed in turn below.

50 S Proteins-Using our improved procedure, the thirty- three 50 S proteins were resolved into at least 22 peaks as shown in Fig. 1. The relative retentions ( a ) of the peaks are listed in Table I. Relative retention is the most reliable index for identification of a species. It is dependent only upon the

~ . I 6

4

.08

0

,004

N a

n

7 5

T I I

4 5 8.

115 " I

2 0 4 0 60 80 I O 0 120 140

E L U T I O N T I M E ( m l n )

FIG. 1. RP-HPLC of TP50. A solution of TP50 (217 pg) in 10 p1 of buffer B was injected onto a Synchropak RP-P column and eluted with a convex gradient (curve 5, Waters 660 Programmer) of 15 to 45% solvent B in 120 min followed by a 10-min isocratic elution at 45% solvent B and a 60-min linear gradient from 45 to 100% solvent B (dashed line). The upper and lower traces are absorbances at 214 nm and 280 nm, respectively. The proteins present in the lettered peaks are listed in Table I.

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RP-HPLC of E. coli Ribosomal Proteins 6315

TABLE I Relative retention of 50 S proteins

- . ~ ~~ .

Peak Protein n o

A L34 R

0.23 L32 0.28

c L33 0.35 D 0.38 E L27 0.46 F L27* 0.55 G 0.62 H 0.69 1 L24 0.78 .I L28 0.84 K L26 L

1 .00 L25 1.03

M L14, L19, LC30 1.17 N L13, L21 1.24 0 L2, L3, L17 1.31 P L18 1.35 Q L22, L23 1.43 K L29 1.48 S L6, L9, L15, L16 1.66 T L11 1.72 U L1 1.78 v L5 1.86 W L20 x

2.21 L4, L10 2.60

Y L12 2.76 z L7 2.79

-~

a Relative retention ( n ) is defined as ( t R , - to) / ( tR, - to) where t ~ , is the retention time of component i and to is the retention time of a nonsorbed solute (solvent front). For the (1 values in Tables I and 11, tR, was chosen as the retention time of L26 (= S20), the only protein common to both subunits. The retention times used to calculate n values in both tables were measured from a TP70 elution profile. The n values reported were reproducible to k0.01.

Peak F contains oxidized L27. When peak F was incubated at 42 "C in buffer B containing 100 mM 2-mercaptoethanol for 2 h, a new peak eluted in the position of L27. The peak also showed a spot corresponding to L27 in two-dimensional PAGE.

. .. ~ ~~

temperature and the composition of the column and the mobile phase, and is independent of column dimensions, instrument dead volume, sample size, and flow rate. The identities of the proteins in the peaks, summarized in Table I, were determined by one or more of the following methods: (a ) one-dimensional PAGE; (b) two-dimensional PAGE; (c) calibration with authentic samples of purified 50 S proteins. In the analyses using purified proteins, relatively high con- centrations of protein standards were applied along with a low concentration of TP50, which served to calibrate the chromatogram. In the two-dimensional PAGE analyses, rel- atively high concentrations of proteins from HPLC peaks were run against a low concentration of background TP30, which served to calibrate the gel. A typical two-dimensional PAGE analysis is presented in Fig. 2, in which peak Q from Fig. 1 is shown to contain proteins L22 and L23. A trace of L18, from the neighboringpeak R, is also seen.

A steep gradient step at the end of the HPLC run was required to elute proteins L4 and L10 (peak X), L12 (peak Y ) , and L7 (peak 2) (Fig. 1). Resolution of the latter two proteins demonstrates that the L7/L12 dimer is disrupted under the elution conditions. Proteins L7/L12 are observed in reduced quantities in TP50 compared to TP70 (see Fig. 5 below). Recovery of 50 S subunits from sucrose gradients by high speed pelleting rather than ethanol precipitation af- forded higher yields of L7/L12 in accord with previous results (3, 34).

All of the previously identified TP50 proteins have been located in the HPLC chromatogram shown in Fig. 1 with the exceptions of proteins L31 and L31', the latter described by

FIG. 2. Identification of proteins by two-dimensional PAGE. Peak Q (Fig. 1) from a TP50 run of approximately 2 mg of total protein was subjected to two-dimensional PAGE as described under "Experimental Procedures." The first dimension was run from k f t to right and the second dimension was run from top to bottom. The spots are due to the proteins present in peak Q plus a background of TP30 which serves to calibrate the gel. Protein identification is according to Kenny et al. (24).

Fanning and Traut (35). These proteins are stained very weakly by Coomassie G-250, and would have been difficult to identify by the PAGE procedures discussed above. Further- more, they have previously been isolated in only small amounts (3). Conversely all of the peaks in Fig. 1 have been identified as containing known ribosomal proteins except for peaks L), G, and H. In contrast to the other peaks in Fig. 1, the relative amounts of peaks D, C, and H varied with condi- tions of TP50 preparation and storage, an indication of their greater susceptibility to degradation. Peak G was the most unstable. When it was lyophilized, redissolved in buffer B, and rechromatographed, no peak eluted in the original posi- tion of peak G . Instead, ten earlier eluting peaks were ob- served. Peaks D and H were unstable to long term storage at -20 "C, but could be stored at -78 "C. On rechromatography of both peaks D and H , the major peaks eluted in the original peak positions, although several minor, earlier eluting peaks were seen with peak D. A composite schematic summary of two-dimensional PAGE runs showing positions due to peaks D and H is shown in Fig. 3. Peak D gave rise to three spots, two of which migrated with the dye front in the area of proteins L32 and L33, and one which was slightly slower in the second dimension. Peak H gave rise to two spots, one migrating near L33 and the other in an area not corresponding to a known ribosomal protein. It remains to be established whether peaks D, G, and H correspond to new 50 S proteins discovered as a consequence of our new HPLC method of analysis, or to degradation products of previously identified 50 S proteins, or to some other procedural artifacts. 30 S Proteins-The improved RP-HPLC procedure de-

scribed above has resulted in an increased resolution of the twenty-one 30 S proteins into 17 peaks (Fig. 4), as compared with the 15 peaks reported earlier (25). The identities of the proteins present in each peak, as summarized in Table 11, were determined by one or more of the following methods: (a ) comparison with the previously standardized TP30 chroma- togram (25); ( b ) calibration with authentic samples of purified 30 S proteins; ( c ) one-dimensional PAGE analysis.

In contrast to the TP50 results, all of the known TP30 proteins have been identified and all of the major peaks

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6316 RP-HPLC of E. coli Ribosomal Proteins

correspond to known 30 S proteins. The smaller unlabeled peaks seen in Fig. 4 are 50 S proteins, present due to minor contamination of our 30 S subunits by 50 S subunits. The intensity of peak e, corresponding to S20, when compared to peak K (L26) (Fig. 1) confirms the previously reported result that this single protein partitions mostly to the 30 S subunit (3). Two further points should be noted. Proteins S1 and S2 were observed in only small amounts reflecting losses during subunit preparation (3), and the shoulder on peak p (arrow, Fig. 4) contains the protein A described by Subramanian et al. (36).

70 S Proteins-The fifty-three 70 S proteins were resolved into at least 31 peaks by our improved RP-HPLC procedure (Fig. 5). The highly reproducible nature of the chromatograms enabled peak assignment to be made largely on the basis of

1st Dimension O R I G I N

4 B O

- S I

e s2 o s 3

o s 4 S 5 @ O S ?

5 6 6 m S 8

s IO. r S 9 , S l l

e s 1 2 S 13-

- s 2 0

s 2 1 . r r( Ly" D D Y E - " " " " + -&H

comparison with the TP30 and TP50 elution profiles. In certain cases, confirmations were obtained using one-dimen- sional PAGE analysis.

A noteworthy point is that the peak eluting at 135 min during the final steep gradient portion of the chromatogram was found by subsequent two-dimensional PAGE analysis to contain several 50 S proteins, in the relative amounts L1, L9 > L4 > L3, L5, L10 > L15. When this peak was lyophilized, redissolved in buffer B, and rechromatographed, greater than 80% of the absorbance at 214 nm eluted in its original posi- tion. Thus, it is likely that this peak contains proteins which are denatured and/or aggregated. It should be emphasized that the amount of any of the proteins found in this peak is small (<lo% even for proteins L1 and L9) compared to the amount eluted in the major peak of that protein as defined in Table I. Further studies designed to understand the basis for this apparent partitioning of some ribosomal proteins into two regions of the chromatogram are underway and will be reported elsewhere.

Resolution of Individual Proteins-With our current pro- cedure 16 of 33 known 50 S proteins, 13 of 21 known 30 s proteins, and 18 of 53 known 70 S proteins are fully resolved in single chromatographic runs of TP50, TP30, and TP70, respectively. It is important to emphasize that, where desira- ble, complete resolution of any given ribosomal protein should be attainable in straightforward fashion by reapplication of

TABLE I1 Relative retention of 30 S proteins -

Peak Protein a'

a s12 0.55 b s21 0.69 C 514 0.82 d s19 0.94 e s20 1.00 f s11 1.03 g S15 1.05 h S18 1.07 1 S17 1.17 j S10, S16 1.38 k S4, S8 1.43 1 S3, S13 1.48 m s5. s9 1.69

FIG. 3. Two-dimensional PAGE analysis of peaks D and H in Fig. 1. A composite schematic was made from individual gels of each peak. Peaks were collected from multiple TP50 runs of approx- imately 12 mp total protein. A background of TP30 was used to

n S6' 1.72 0 s 7 1.78 P S2, A 2.11 9 s1 2.60

calibrate the gel.

FIG. 4. RP-HPLC of TP30. TP30 (84 pg) was dissolved in 15 pl of buffer B, injected onto a Synchropak RP-P col- umn, and eluted with a convex gradient (curve 5, Waters 660 Programmer) of 15 to 45% solvent B in 120 min, followed by an additional 20-min isocratic elution at 45% solvent B (dashed line). The up- per and lower traces are absorbances a t 214 nm and 280 nm, respectively. The proteins present in the letteredpeaks are listed in Table 11. The arrow corresponds to protein A described by Subramanian et al. (36).

See Footnote a in Table I.

rn

.o 8

N

a .o 4

0

,002 m N

a 0

2 0 40 60 80 I O 0 I 2 0

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RP-HPLC of E. coli Ribosomal Proteins 6317

FIG. 5. RP-HPLC of TP70. TP70 (442 pg) was dissolved in 16 p1 of buffer B, injected onto a Synchropak RP-P col- umn, and eluted with a gradient identical with that in Fig. 1 (dashed line). The upper and lower truces are absorbances at 214 nm and 280 nm, respectively. The upper case letters refer to the TP50 pro- teins in Table I and the lower case Zetters refer to the TP30 proteins in Table 11. The arrow corresponds to a collection of small amounts of several TP50 proteins (see text).

.o 2 4 N

a

0

.o 0 0

a : o

.I 6

.I 2

a .oe

. 0 4

C

,002

U C

L I 9

0

I

i

n

;I I 1 I I I 1 I

20 40 60 80 100 120 140 E L U T I O N TIME ( m i n )

7 5

T - 1

m

,\" 4 5

15

I I I I 1

I O 30 5 c ELUTION TIME ( m i n )

FIG. 6. Resolution of peak M (Fig. 1). Protein (-70 pg) eluting in peak M (Fig. 1) was lyophilized, redissolved in 10 pl of buffer B, and injected onto a Synchropak RP-P column equilibrated at 35% solvent B. After 15 min at 35% solvent B, a linear gradient was run from 35% solvent B to 40% solvent B in 60 min (dashed line). The upper and lower truces are absorbances at 214 nm and 280 nm, respectively.

an unresolved peak to the column and re-elution with a shallower gradient. For example, peak M from Fig. 1, contain- ing proteins L14, L19, and L30, was resolved into three peaks (Fig. 6) when reapplied to the column at 35% solvent B and eluted with a 60-min linear gradient to 40% solvent B. The proteins were identified by one-dimensional PAGE. In cases where elution with a shallower gradient does not give full resolution, a column having a functional group with a polarity different than C18-silica (e.g. NH2-, CN-, or phenyl-silica), might be employed in the second step.

Functionality of HPLC-purified Proteins-The results al- ready presented make clear the utility of the RP-HPLC technique for analysis of ribosomal proteins. A separate ques- tion concerns the utility of this method for preparation of proteins which could be used in functional studies. The su- crose density gradient results presented in Fig. 7 provide a clear demonstration that HPLC-purified proteins are capable of being reconstituted into 30 S subunits on recombination with 16 S RNA. The functionality of these subunits was tested

0 W N

a

30 s 1

1 , 1 , , 1 1 , , , , 1 1 ,

8 16 24 V O L U M E ( r n l )

FIG. 7. Sucrose gradient centrifugation of reconstituted and native 30 S particles. Fractionation profiles for 36-ml sucrose gradient runs for native 30 S subunits (-), subunits reconstituted from acetic acid-extracted proteins (- - -), and subunits reconstituted from acetic acid-extracted proteins which had been eluted from a Synchropak RP-P column (-.-) are shown. The direction of sedi- mentation is from left to right and the volume is given in milliliters from the top of the gradient.

TABLE 111 Effect of HPLC treatment on Phe, tRNAp" binding

Sample Phe . tRNAPhe binding assay

Relative

Experi- 30 S Extrac- HPLC phe, poly(u)- merit sub- tion treat- Poly(U) tRNAPhe

unit" method* ment bound tRNAPhe

binding

% 1 N 5.4 2 N + 67.2 3 R AA + 21.6 0.26

1.00

4 R AA + + 18.6 0.21 5 R LCU - + 16.0 6 R LCU + + 13.2 0.13

0.17

- - - -

N, native; R, reconstituted. * AA, acetic acid; LCU, LiC1-urea.

by their ability to bind Phe. tRNAPh" in a poly(U)-dependent manner. The activity of reconstituted 30 S subunits prepared from proteins extracted using either the acetic acid (16)- or LiC1-urea (17) procedures, and then subjected to HPLC treat-

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6318 RP-HPLC of E. coli

ment, were compared with the activities of reconstituted 30 S subunits from extracted proteins not subjected to HPLC treatment. The results, presented in Table 111, show only a minor loss in activity (<25%) as a result of HPLC treatment. The relatively low Phe . tRNAPhe binding for reconstituted 30 S subunits compared to native 30 S subunits was most likely due to partially degraded 16 S RNA.

CONCLUSION

The results presented in this paper establish RP-HPLC as the method of choice for both the analysis and preparation of E. coli ribosomal proteins. By comparison with the methods previously used for these purposes, polyacrylamide gel elec- trophoresis for analysis and either ion exchange or size exclu- sion chromatography for preparation, RP-HPLC offers at least equivalent resolution and reproducibility, and is clearly superior with respect to rapidity, recovery yields, and, with the use of the 214 nm monitor, sensitivity. In our own labo- ratory, we are currently making extensive use of the HPLC technique both to analyze proteins from photoaffinity-labeled ribosomes,z and to prepare proteins for reconstitution exper- iments. Extension of the RP-HPLC method to study eucar- yotic ribosomal proteins should be straightforward. I t may also be useful in studying othe? complex cellular components having heterogeneous protein compositions.

Acknowledgments-We wish to acknowledge the superb technical skills of Nora Zutio and Anthony Nixon who assisted in several aspects of this work, and the gifts of purified ribosomal proteins from Drs. Nomura (University of Wisconsin) and Dahlberg (Brown Uni- versity).

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J . Biol. Chem. 249, 3103-3111 5. Hampl, H., Schulze, H., and Nierhaus, K. H. (1981) J. Bid.

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8. Cooperman, B. S. (1978) Bioorg. Chem. IV, 81-115 9. Cooperman, B. S. (1980) in Ribosomes (Chambliss, G., Craven,

C . J. Weitzmann and B. S. Cooperman, manuscript in prepara- tion.

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10. Kuechler, E., and Ofengand, J. (1980) in Transfer RNA: Struc- ture, Properties, and Recognition (Schimmel, P., Soll, D., and Abelson, J., eds) pp. 413-444, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

11. Moore, P. B. (1980) in Ribosomes (Chambliss, G., Craven, G. R., Davies, J., Davis, K., Kahan, L., and Nomura, M., eds) pp. 111-133, University Park Press, Baltimore

12. Nierhaus, K. H. (1982) Curr. Top. Microbiol. Zmmunol. 97, 82- 155

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A R Kerlavage, T Hasan and B S Coopermanchromatograms.

ribosomal proteins: standardization of 70 S, 50 S, and 30 S protein Reverse phase high performance liquid chromatography of Escherichia coli

1983, 258:6313-6318.J. Biol. Chem. 

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