Ribonucleic Acid of Escherichia coli

Transcription

1 JOURNL OF BTERIOLOGY, JUlY 1973, p opyright merican Society for Microbiology Vol. 115, No. 1 Printed in lj.s.. Localization of a Binding Site for Ribosomal Protein S8 Within the 16S Ribosomal Ribonucleic cid of Escherichia coli H. W. SHUP, M. L. SOGIN,. G. KURLND,' ND. R. WOESE Department of Microbiology, University of Illinois, Urbana, Illinois Received for publication 31 March 1973 region of 16S ribosomal ribonucleic acid that binds Escherichia coli ribosomal protein S8 has been isolated and characterized. It corresponds to what has been designated as fragment of the ribonucleic acid. Five 30S ribosomal proteins can individually bind to specific sites on the 16S ribosomal ribonucleic acid (rrn) under simple, controlled conditions in vitro (12, 13). Earlier work described how such RN-protein complexes can be degraded with nuclease to localize the binding site for a given ribosomal protein (14, 15). Our data for the RN region (called S4aR) which binds protein S4 indicate that this protein may bind to a number of sites on the RN. Furthermore, S4aR is likely to have rather substantial base paired regions (5). These observations and the large size of S4aR suggest that S4 can alter the conformation of the RN when it forms the site-specific complex. Such induced conformational changes are expected to influence the binding of other proteins. In this report we localize a binding site for 30S ribosomal protein S8. In an earlier communication we presented evidence that protein S8 will bind to intact 16S rrn in the absence of other ribosomal proteins (12). lthough this binding is not stoichiometric (0.65 moles of protein S8 per mole of 16S rrn), the addition of proteins S4 and S8 makes it possible to bind one molecule of S8 to every 16S rrn molecule (12). The association of S8 with rrn is specific in the sense that no other 30S protein will compete with it for its "binding site" on the intact 16S rrn (12). In view of the'cooperative relationship between S8 and S4 we conclude, from the evidence presented below, that S8 binds to an RN site (S8aR) located near S4aR. MTERILS ND METHODS Proteins and rrn, labeled and unlabeled, were prepared as previously described from Escherichia coli B236 (6, 12). Tritiated L-lysine (104-mi/mmole; Schwarz/Mann) was used in the culture media to label ribosomal proteins. By following previously detailed procedures for purification of 30S protein S88 3H-S8 was obtained with a specific activity of counts per min per,g and a 28% counting efficiency for tritium. The proteins prepared by these procedures are greater than 95% pure by electrophoretic analysis (6). The RN was either labeled with 14uracil (800 mi/mmole; Schwarz/Mann) or carrierfree 32P-orthophosphate. The '4-rRN specific activity at 63% counting efficiency was 100 counts per min per g and 32P-rRN was 2 x 106 to 5 x 101 counts per min per g at greater than 90%c counting efficiency. Purification of rrn by polyacrylamide gel electrophoresis was employed (1, 7, 16). RN was isolated from polyacrylamide gel slices by direct phenol extraction (2). These RN preparptions were determined by mass to contain less than 2% protein, and whatever protein there is, is not (by polyacrylamide gel analysis) of the ribosomal protein type (Schaup, unpublished data). Protein concentration was determined by the method of Lowry et al. (8), and rrn mass was ascertained spectrophotometrically by using an E260mm of 227 (9). Production of protein-rn complexes, their digestion with ribonuclease, and isolation of the resulting complexes by sucrose density gradient centrifugation have been described (14). Biogel P-30 obtained from Biorad, Inc. was equilibrated over night at room temperature in the ribosome reconstitution buffer described by Traub and Nomura (17). Protein was removed from ribonucleoprotein complexes by three phenol extractions with water-saturated redistilled phenol in the presence of 0.04% sodium lauryl sulfate (15). For two-dimensional electrophoretic "fingerprint" analysis, the purified RN was dialyzed into H20. The nucleic acid fingerprinting was performed by the method of Sanger et al. (11, 16; S. Sogin, Ph.D. thesis, Univ. of Illinois, Urbana, 1970). RESULTS IPresent address: The Wallenberg Laboratory, The Uni- The specific complex between 30S ribosomal versity of Uppsala, Sweden. protein S8 and the 16S RN is formed when the 82

2 VOL. 115, 1973 BINDING SITE FOR E. OLI RIBOSOML PROTEIN S8 83 two are incubated together under the usual conditions for in vitro reconstitution of the 30S subunit (10, 12, 13). Treatment of this S8-rRN complex in reconstitution buffer with 1,ug of pancreatic ribonuclease per 50 jg of complexed RN for 5 min at 22 followed by fractionation on a Biogel P-30 column permits recovery of the putative S8-rRN particle (Fig. 1). The figure shows the following. (i) Nuclease digestion of the S8-rRN mixture yields an RN elution profile that is characteristic of the complex; i.e., digestion of RN alone under these conditions does not yield large amounts of material in the early fractions, 10 to 17. (ii) Protein S8 is eluted differently in the presence and absence of RN. In the presence of RN most of the protein that would be expected to be associated with the RN is eluted in fractions 14 to 17 (with about 15 to 20% of the total RN applied to the column). In the absence of RN, protein recoveries from the column are rather poor, usually less than 10% of the total applied, and the recoverable protein is found in later fractions, 18 to 26. It should also be noted that digestion of the S8-RN mixture produces some large RN fragments (eluting in fractions 10 to 13) that do not appear to be associated with protein. Definitive proof that we are dealing here with a bona fide S8-RN complex (S8aR) can be seen in Fig. 2. In the absence of RN the protein sediments very slowly through sucrose gradients (Fig. 2). However, the putative S8-RN complex isolated from Biogel columns sediments far more rapidly (Fig. 2B,, D). What these data do not prove is that all of the RN is associated with protein. ertainly, this is not the case in Fig. 2B and. It should be noted also that RN isolated from this complex, when mixed with protein S8 under the same conditions, forms a protein-rrn complex which sediments more rapidly than protein alone (Fig. 3). The zone where this complex is located appears to be comparable to that shown in Fig. 2 and D. It is not possible to calculate the stoichiometry of S8 association or reassociation with its RN binding region because we have not accurately ascertained the molecular weight of the RN fragment involved. crylamide-sodium dodecyl sulfate gel electrophoresis of the RN separated from the protein have not yielded sharp bands characteristic of a homogeneous preparation of RN, which suggests that we are possibly dealing with a heterogeneous collection of fragments which may not all be capable of associating with the protein. The profiles seen in Fig. 1 and 2 are highly reproducible and to date are unique for the treated S8rRN complex. For example, protein S4 yields a complex under these conditions that is eluted from the column mainly in fractions 10 to 13, and which sediments more rapidly in a sucrose gradient (14). In a similar experiment in which high specific activity 32P labeled RN was used to make the complex, the centrifugal fractions containing the S8-RN complex.(no in Fig. 2) were isolated, phenol extracted, dialyzed into water, and fingerprinted by two-dimensional electro- E x2103x1x103 B 103_ X103 z~~~~~~~~~ L) I~~~~~~~~~~~~~~~~ FRTION NUMBER FIG. 1. Biogel P-30 column elution profile at 4. The column dimensions were 0.4 cm in diameter and 50 cm high with a flow rate of 4.5 ml/h, and the fraction size was 8 ml. The buffer was ribosome reconstitution buffer (17). Sample application volume was 200X and was prepared as described in the text below., Elution profile for 16S rrn (-, specific activity 100 counts per min per jg) protein S8 (0, specific activity 1,800 counts per min per,g) complex, prepared as described in Methods and Materials. B, Elution profile for rrn alone treated similarly., Elution profile for protein S8 alone; arrow indicates the elution position of the putative proteinrrn complex. E U, Z

3 84 SHUP ET L. J. BTERIOL B E E c. < o300 D z LII ck z~~~~~~~~~~~~~~~~~~~~~~~( FI.2 oecnrfgto fsa eie rma iglp3 oun eaainwspromdo Bckman SW0.Doo 100 at5,0 p o.th rdetws25t 2 ioulaefe scoe(cwr/an FRTION NUMBER FIG. 2. Zone centrifugation of S8aR derived from a Biogel P-30 column. Separation was performed on a Beck- 5 W5 rotor at 50,001K rpm for 9 h. The gradient was 2.5 to 20%/ ribonuclease-free sucrose (Schwarz/Mann) Iman iin ribosome reconstitution buffer (17). Thbes were fractionated by puncturing the bottom and collecting 18O ifractions. Fraction 1 represents the top of the gradient., Protein S8 alone; B,, and D, Biogel P-30 column fractions 14, 15, and 16, respectively (see Fig. 1). j B 600 -c Ea. z al FRTION NUMBER FIG. 3. Reassociation of protein S8 with purified S8aR. The S8-S8aR ribonucleoprotein complex was produced as described in Materials and Methods and isolated by chromatography on Biogel P-30. The RN from pooled column fractions 14 to 16 was phenol extracted. Purified RN was then incubated with ribosomal protein S8, and the mixture was layered on a sucrose density gradient (2.5-20%o sucrose in reconstitution buffer) and sedimented as described in Fig. 2 at 50,000 rpm for 9 h in an SW5 rotor. Fraction I represents the top of the gradient., Protein S8 alone; B, S8aR RN alone;, protein S8 with S8aR. Symbols: open squares, protein (13H); closed circles, RN (14).

4 VOL. 115, 1973 BINDING SITE FOR E. OLII RIBOSOML PROTEIN S8 85 phoresis (11, 15, 16). Table 1 shows the T, nuclease oligomer catalogue obtained from the S8-associated RN (S8aR). The quantities were normalized to a unit mole presence of the oligomer UUG. The corresponding oligomer catalogue generated by pancreatic nuclease is shown in Table 2. DISUSSION We have reported above and elsewhere that ribosomal protein S8 forms a specific complex with the 16S RN (12). The data presented here indicate that after nuclease digestion, the protein remains bound to an RN fragment(s), and that protein S8 will reassociate with such a fragment under conventional in vitro ribosomal reconstitution conditions (17). What we have yet to demonstrate is whether all of the RN present in our above "binding site" fraction is indeed associated with protein S8. In other words, the procedures used to date do not necessarily yield the binding portion of the RN, S8aR, in a high state of purity. ertainly, the larger oligomers present in relatively high G G G UG TBLE 1. Major oligomers (> 0.5 molar ratio) T, nuclease oligomers present in S8aRa Minor oligomers ( <0.5 molar ratio) Oligomer Occurrence T Location Oligomer [ Occurrence T Location G G G UG UG UG UG UUG (U2, ) G UG UG UUUG U... UUG UUG UUG UUUG Elor K, or ' Lor 0, G G G G UG UUG UUG *GG G sg sg UG UG UG UG UG UG UG UG OUG su4g (U, 2, 2)G UUG UUG UUG (U2, )G (2, U)UG (, US)G... U3G (U, 4)UUG (2, U2)USG " M or p F L H' Q-Q' ' ' K ord aribonuclease T, oligomer catalogue for S8aR. Oligomers are separated into high-occurrence and lowoccurrence groups. The composition and relative occurrence are given under those headings based on unit mole presence of UUG for each oligomer in S8aR. The location of the oligomer within previously sequenced fragments of 16S rrn are indicated by the letter under "location." From the 5' terminus of the 16S rrn the order of the fragments indicated above is as follows: L, *, Q, Q', F, H', *,., M, *, ",, ', 0, D, E', P, K,... The. indicates intervening segments not found in S8aR preparations (5). E' F

5 86 SHUP ET L. J. BTERIOLI. molar ratios (Table 1 and 2) must, by all accounts, represent true S8aR. Some of the oligomers present in lower molar ratios also represent S8aR. However, we cannot decide, on the basis of the present studies, whether TBLE 2. Pancreatic nuclease oligomers presenta Major oligomers Minor oligomers (>0.6 molar ratio) (<0.5 molar ratio) Oligomer Occurrence Oligomer Occur- rence U 18 U 11 GU U G 0.5 GU 5.9 (G, G) 0.5 G 5.2 GGU 0.4 GGG U - G 1.7 (G, G)U G 1.1 GU 2.5 G GG 2.6 GU GGU 2.5 (G, G)U (G, G)U 0.5 G 1.1 (G, G2)U GU 1.3 GGGGU 0.4 GU 1.9 GG 0.6 GG GGGU 0.7 (2G, G) (G, G) GU 0.6 4G2GU 0.4 (G, G4)U (2G, G2) G3 - (G, G3)U (G, Gs)U (G4, G)U Pancreatic ribonuclease oligomer catalogue for S8aR. Oligomer sequence and relative occurrences are given. this is true for the remaining low molar ratio oligomers. It should be noted that the low occurrence oligomers do not represent a nonspecific background, in that most of the T, oligomers characteristic of the 16S rrn are not detectable in digests of our S8aR preparations. The above sedimentation analyses and oligomer catalogues show S8aR to be smaller than its counterpart, S4aR, which comprises about 30% of the total 16S rrn molecule (14). S8aR seems to correspond closely to a region approximately 700 bases from the 5' terminus of the 16S rrn molecule which has been desi,nated as fragment (Fig. 4; references 5. 18). ll but one of the larger (i.e., unique) T, oligomers found in high yield in S8aR preparations are located in fragment. lthough it is less striking, the same situation pertains for the olig,omers produced by pancreatic nuclease. Figure 4 shows an interesting feature of S8aR; the loop and the adjcent portion of the presumably double-stranded stalk found in fragment are absent in S8aR and so are not protected from nuclease attack during S8aR preparation. Note too that the T, oligomer catalogue (Table 1) contains the artificially produced 3' termiinal oligomer, U, derived from the large T, oligomer, UG, by pancreatic nuclease cleavage of fragment. It is possible that S8aR extends to one or both sides of frament ""; for several large T, oligomers from fragment ' (adjacent to on the 3' end) are found in S8aR preparations in about a mole ratio (Table 1). S4aR covers the contiguous segments of the 16S rrn molecule called F, H', H, G. NI. B. I", and I (5, 13). Fragment "," the main component of S8aR, is located nearby (in terms of the primary structure), being onlv some 100- odd nucleotides distant (3, 5). Thus, the coop ~ U G GUUUGUU GU G UG U GU G G G li I I I I I I I I I I 3' G U U G G GUU G U U G U() G G G U U 5 < FIG. 4. The reported sequence of fragment "," showing its presumed secondary structure (3). Solid lines show T1 nuclease oligomers found in S8aR and their relative occurrence therein; dotted lines show pancreatic nuclease oligomers, etc. The T, oligomer UUG contained in the loop of fragment is not found at detectable levels in S8aR. The two oligomers UG and UG are reported to be alternative forms (5); their joint occurrence, i.e., the representation of this region in S8aR, is + = 0.4.

6 VOL. 115, 1973 BINDING SITE FOR E. OLI RIBOSOML PROTEIN S8 erative effect reported for protein S4 on protein S8 binding to rrn can be readily understood (12). It has been reported elsewhere that fragment binds ribosomal protein S15, although no binding of S8 by fragment is mentioned (19). This and the present finding are not necessarily contradictory. Further scrutiny of this matter is definitely required because the possible binding of two ribosomal proteins to so small an RN segment would certainly generate a host of interesting phenomena when studied in detail. KNOWLEDGMENTS This work was supported by Public Health Service grant I-6457 from the National Institute of llergy and Infectious Diseases to.r.w. H.W.S. was the recipient of a Public Health Service postdoctoral fellowship. LITERTURE ITED 1. Bishop, D. H. L., J. R. laybrook, and S. Spiegelman Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J. Mol. Biol. 26: ory, S., J. M. dams, P. Spahr, and U. Rensing Sequence of 51 nucleotides at the 3'-end of R17 bacteriophage RN. J. Mol. Biol. 63: Ehresmann,., J. L. Fischel, P. Fellner, and J. P. Ebel Studies on the primary structure of the 16S ribosomal RNs of Escherichia coli and proteus vulgaris. Seventh FEBS Meeting, Varna 23: Fellner, P.,. Ehresmann, and J. P. Ebel Nucleotide sequences present within the 16S ribosomal RN of Escherichia coli. Nature (London) 225: Fellner, P.,. Ehresmann, P. Stiegler, and J. P. Ebel Partial nucleotide sequence of 16S ribosomal RN from E. coli. Nature N. Biol. 239: Hardy, S. J. S.,. G. Kurland, P. Voynow, and G. Mora The ribosomal proteins of Escherichia coli. I. Purification of the 30S ribosomal proteins. Biochemistry 8: Hecht, N. B., and. Woese Separation of bacterial ribosomal ribonucleic acid from its macromolecular precursors by polyacrylamide gel electrophoresis. J. Bacteriol. 95: Kurland,. G Molecular characterization of ribonucleic acid from Escherichia coli ribosomes. I. Isolation and molecular weights. J. Mol. Biol. 2: Lowry, 0. H., N. J. Rosebrough,. J. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. hem. 193: Nomura, M., S. Mizushima, M. Ozaki, P. Traub, and. V. Lowry Structure and function of ribosomes and their molecular components. old Spring Harbor Symp. Quant. Biol. 34: Sanger, F., G. G. Brownlee, and B. G. Barrell. two-dimensional fractionation procedure for radioactive nucleotides. J. Mol. Biol. 13: Schaup, H. W., M. Green, and. G. Kurland Molecular interactions of ribosomal components. I. Identification of RN binding sites for individual 30S ribosomal proteins. Mol. Gen. Genet. 109: Schaup, H. W., M. Green, and. G. Kurland Molecular interactions of ribosomal components. II. Site-specific complex formation between 30S proteins and ribosomal RN. Mol. Gen. Genet. 112: Schaup, H. W., and. G. Kurland Molecular interactions of ribosomal components. III. Reconstitution of a ribosomal protein nucleic acid binding site. Mol. Gen. Genet. 114: Schaup, H. W., M. Sogin,. Woese, and. G. Kurland haracterization of an RN "binding site" for a specific ribosomal protin of Escherichia coli. Mol. Gen. Genet. 114: Sogin, M., B. Pace, N. R. Pace, and. R. Woese Primary structural relationship of P16 to M16 ribosomal RN. Nature (London) 232: Traub, P., and M. Nomura Structure and function of Escherichia coli ribosomes. V. Reconstitution of functionally active 30S ribosomal particles from RN and protein. Proc. Nat. cad. Sci. U.S.. 59: Zimmerman, R..,. Muto, P. Fellner,. Ehresmann, and. Branlant Location of ribosomal protein binding sites on 16S ribosomal RN. Proc. Nat. cad. Sci. U.S.. 69: