Translation initiation complex formation with 30 S ribosomal particles mutated at conserved positions in the 3'-minor domain of 16 S RNA.

Escherichia coli 30 S ribosomal subunits containing in vitro (phage T7 RNA polymerase-generated) 16 S rRNA, both wild-type and mutant, were examined by toeprinting. These synthetic particles were used to compare the effects of the absence of base modification and of specific nucleotide substitutions in conserved sequence regions of the RNA on the assembly of mRNA, tRNAs and 30 S particles into a translational initiation complex. Initiation factor-3-dependent selection of tRNA(fMet) from a mixture of tRNA(fMet) and tRNA(Phe) occurred with all particles, although 20 times less initiation factor-3 was needed for the synthetic particles, including the mutants. Whereas isolated 30 S particles or those reconstituted with isolated RNA did not distinguish between tRNA(fMet) and tRNA(Phe) for ternary complex formation in the absence of initiation factor-3 (intrinsic selection ability), the synthetic particles preferred tRNA(fMet). The difference between the natural and synthetic particles appears to be due to the absence of certain base modifications, but not m2(6)A, in the synthetic RNA. Synthetic particles containing the mutation U1512C, which converts the universal U.G pair to C.G enhanced both tRNA(fMet) binding and selectivity, although other mutations at that site, namely U1512G, G1523A and U1512C/C1524U, had no such effect. Mutants U1498G and G1401C/C1501G, both located in a highly conserved single-stranded region of the 3'-minor domain, also enhanced tRNA(fMet) selectivity, in this case by reducing complex formation with elongator tRNA. Complex formation between elongator tRNA and the G1401C/C1501G mutant was reduced to almost undetectable levels. The results also indicated that the association rate for initiation complex formation for G1401C/C1501G was considerably lower than for the wild-type sequence. This result had not been detected by standard tRNA-30 S binding assays. Overall, the data suggest that (some of) the 16 S rRNA base modifications as well as the tertiary structure around the decoding site act to desensitize the intrinsic selection ability of the ribosome for tRNA(fMet).

Functional effects of base changes which further define the decoding center of Escherichia coli 16S ribosomal RNA: mutation of C1404, G1405, C1496, G1497, and U1498.

The existence and functional importance of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, was proven recently by mutational analysis [Cunningham, P. R., Nurse, K., Bakin, A., Weitzmann, C. J., Pflumm, M., & Ofengand, J. (1992) Biochemistry 31, 12012-12022]. Here we show that the additional nearby tertiary base pairs C1404:G1497 and G1405:C1496 also exist and are functionally important for tRNA binding to the ribosomal A and P sites. Breakage of the base pairs in turn led to a loss of activity at both A and P sites, whereas restoration in the reverse orientation led to recovery of activity. Recovery was incomplete, indicating that base pairing alone is insufficient for full restoration of function. Mutation of U1498 to G created the potential for the tertiary base pair C1403:G1498, which could stack on the aforementioned double base pair, creating a more stable helix longer by one residue. This mutation did not affect subunit association, A- and P-site binding of tRNA to 70S, fMet-tRNA binding to 30S, or poly(Phe) synthesis but did block formation of the first peptide bond, fMet-Val. Mutation of U1498 to A or C did not show this effect. Since the G1498 mutant could make both the 70S initiation complex and the peptide bond, as shown by its ability to form fMet-puromycin, the block in fMet-Val synthesis appears to involve some aspect of A-site function.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical evidence for domain assembly of the Escherichia coli 30S ribosome.

A fragment of 16S RNA corresponding to most of the 5'-domain (residues 1-526) was prepared by in vitro run-off transcription. When this fragment was incubated with a mixture of 30S proteins under conditions known to result in the in vitro assembly of a complete, functional 30S ribosome from a full-length transcript, a discrete 16S particle was formed. This particle contained near stoichiometric amounts of ribosomal proteins S4, S16, S17, and S20. These four proteins are the same, and only, ones that have been shown to interact with the 5' domain of 16S RNA in the intact 30S ribosome in the footprinting studies of Noller and co-workers. We conclude that the 5' fragment 1-526 is capable of folding independently of the rest of the molecule so as to generate the protein binding sites for the same four proteins with which the corresponding segment of full-length 16S RNA normally interacts. These sites not only include those for S4, S17, and S20 that are known to bind directly to the RNA, but also the site for S16, which requires the prior binding of S4 and S20.

Interaction between the two conserved single-stranded regions at the decoding site of small subunit ribosomal RNA is essential for ribosome function.

Formation of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, is essential for ribosome function. It was previously reported that mutation of G1401 inactivated all in vitro functions of the ribosome [Cunningham et al. (1992) Biochemistry 31, 7629-7637]. Here we show that mutation of C1501 to G was equally inactivating but that the double mutant C1401:G1501 with the base pair reversed had virtually full activity for tRNA binding to the P, A, and I sites and for peptide bond formation. Initiation-dependent formation of the first peptide bond remained 70-85% inhibited, despite full 70S initiation complex formation ability as evidenced by the ability to form fMET-puromycin. These results suggest that the defect in formation of the first peptide bond lies in filling the initial A site, Ai, rather than the subsequent elongation A sites, Ae. An increased mobility around the anticodon was detected by UV cross-linking of the anticodon of P-site-bound tRNA to C1399 as well as to the expected C1400. These findings provide the first experimental evidence for the existence of the G1401:C1501 base pair and show that this base pair, located at the decoding site, is essential for function. The structural implications of tertiary base pair formation are discussed.

G1401: a keystone nucleotide at the decoding site of Escherichia coli 30S ribosomes.

16S ribosomal RNA contains three highly conserved single-stranded regions. Centrally located in one of these regions is the C1400 residue. Zero-length cross-linking of this residue to the anticodon of ribosome-bound tRNA showed that it was at or near the ribosomal decoding site [Ehresmann, C., Ehresmann, B., Millon, R., Ebel, J-P., Nurse, K., & Ofengand, J. (1984) Biochemistry 23, 429-437]. To assess the functional significance of sequence conservation of rRNA in the vicinity of this functionally important site, a series of site-directed mutations in this region were constructed and the effects of these mutations on the partial reactions of protein synthesis determined. Mutation of C1400 or C1402 to any other base only moderately affected a set of in vitro protein synthesis partial reactions. However, any base change from the normal G1401 residue blocked all of the tested ribosomal functions. This was also true for the deletion of G1401. Deletion of C1400 or C1402 had more complex effects. Whereas subunit association was hardly affected, 30S initiation complex formation was blocked by deletion of C1400 but much less so by deletion of C1402. Alternatively, tRNA binding to the ribosomal A site was more strongly affected by deletion of C1402 than by deletion of C1400. P site binding was inhibited by either deletion. HPLC analysis of the in vitro reconstituted mutant ribosomes showed that none of the functional effects were due to the absence or gross reduction in amount of any ribosomal protein.(ABSTRACT TRUNCATED AT 250 WORDS)

A paradigm for local conformational control of function in the ribosome: binding of ribosomal protein S19 to Escherichia coli 16S rRNA in the presence of S7 is required for methylation of m2G966 and blocks methylation of m5C967 by their respective methyltransferases.

We have partially purified two 16S rRNA-specific methyltransferases, one of which forms m2G966 (m2G MT), while the other one makes m5C967 (m5C MT). The m2G MT uses unmethylated 30S subunits as a substrate, but not free unmethylated 16S rRNA, while the m5C MT functions reciprocally, using free rRNA but not 30S subunits (Nègre, D., Weitzmann, C. and Ofengand, J. (1990) UCLA Symposium: Nucleic Acid Methylation (Alan Liss, New York), pp. 1-17). We have now determined the basis for this unusual inverse specificity at adjacent nucleotides. Binding of ribosomal proteins S7, S9, and S19 to unmodified 16S rRNA individually and in all possible combinations showed that S7 plus S19 were sufficient to block methylation by the m5C MT, while simultaneously inducing methylation by the m2G MT. A purified complex containing stoichiometric amounts of proteins S7, S9, and S19 bound to 16S rRNA was isolated and shown to possess the same methylation properties as 30S subunits, that is, the ability to be methylated by the m2G MT but not by the m5C MT. Since binding of S19 requires prior binding of S7, which had no effect on methylation when bound alone, we attribute the switch in methylase specificity solely to the presence of RNA-bound S19. Single-omission reconstitution of 30S subunits deficient in S19 resulted in particles that could not be efficiently methylated by either enzyme. Thus while binding of S19 is both necessary and sufficient to convert 16S rRNA into a substrate of the m2G MT, binding of either S19 alone or some other protein or combination of proteins to the 16S rRNA can abolish activity of the m5C MT. Binding of S19 to 16S rRNA is known to cause local conformational changes in the 960-975 stem-loop structure surrounding the two methylated nucleotides (Powers, T., Changchien, L.-M., Craven, G. and Noller, H.F. (1988) J. Mol. Biol. 200, 309-319). Our results show that the two ribosomal RNA MTs studied in this work are exquisitely sensitive to this small but nevertheless functionally important structural change.

SP6 RNA polymerase stutters when initiating from an AAA... sequence.

The 16S ribosomal RNA gene of Escherichia coli was placed under the transcriptional control of consensus and modified T7 promoters and a modified SP6 promoter. Both T7 and SP6 polymerases faithfully transcribed the coding sequence (beginning at the +1 position) of each construct, although SP6 polymerase was five-fold more effective than T7 polymerase in initiating with the AAAUUG... sequence. An appreciable fraction of the SP6 transcript molecules contained additional adenosines in the -1, -2, -3, -4, and -5 positions. The transcripts containing additional residues constituted approximately 40-50% of the total SP6 transcription products. Neither the nature nor extent of the additional residues was affected by replacing the pppA 5'-end by pA. Since the identity of the inserted residues does not correspond to the sequence of the template, these additional nucleosides must result from 'stuttering' of the SP6 enzyme at the -1 to +3 positions during initiation of transcription.

The absence of modified nucleotides affects both in vitro assembly and in vitro function of the 30S ribosomal subunit of Escherichia coli.

16S RNA of Escherichia coli lacking all post-transcriptional modifications and with 5'-termini of pppGGGAGA-, pppGAA-, pppAAA-, and pAAA- were prepared by in vitro transcription of appropriately engineered plasmids with T7 or SP6 RNA polymerases. These synthetic versions of 16S RNA were compared with natural 16S RNA for their ability to reconstitute 30S ribosomal subunits in vitro using varied conditions for both the isolation of the RNA and for reconstitution. Under all conditions studied, natural 16S RNA assembled correctly, as judged by velocity centrifugation comparison with an internal standard of native 30S particles, and the recovered ribosomes were 80-100% as active as native 30S ribosomes in initiation complex formation, P site binding of AcVal-tRNA, A site binding of Phe-tRNA, and formation of the first peptide bond. In contrast, all of the synthetic constructs including pAAA-, which has the same sequence as native 16S RNA, were only partially active in reconstitution and in the functional assays. We conclude that the lack of the 10 methylated nucleotides and/or the 2 pseudouridylate residues present in natural 16S RNA must be responsible for the reduced activity of the synthetic RNAs in ribosome assembly and function.

Site-specific mutation of the conserved m6(2)A m6(2)A residues of E. coli 16S ribosomal RNA. Effects on ribosome function and activity of the ksgA methyltransferase.

In vitro synthesis of mutant 16S RNA and reconstitution with ribosomal proteins into a mutant 30S ribosome was used to make all possible single base changes at the universally conserved A1518 and A1519 residues. All of the mutant RNAs could be assembled into a ribosomal subunit which sedimented at 30 S and did not lack any of the ribosomal proteins. A series of in vitro tests of protein synthesis ability showed that all of the mutants had some activity. The amount varied according to the assay and mutant, but was never less than 30% and was generally above 50%. Therefore, neither the conserved A1518 nor A1519 residues are essential for ribosome function. The mutant ribosomes could also be methylated by the ksgA methyltransferase to 70-120% of the expected amount. Thus, neither of the A residues is required for methylation of the other, ruling out any obligate order of methylation of A1518 and A1519.

Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA.

The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence. The length and sequence of the 3' terminal T1 fragment was also as expected from the DNA sequence, except that an additional C, A, or U residue was added to 21%, 18%, or 5% of the molecules, respectively. Typical transcription reactions yielded 500-700 moles RNA per mole template. This transcript was used as a substrate for methyl transfer from S-adenosyl methionine catalyzed by Escherichia coli cell extracts. The majority (50-65%) of activity observed in a crude (S30) extract appeared in the post-ribosomal supernatant (S100). Activities catalyzing formation of m5C, m5U, m2G, and m6A residues in the synthetic transcript were observed.

Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences.

In previous work we have shown that both puromycin [Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274] and p-azidopuromycin [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Coooperman, B. S. (1982) Biochemistry 21, 3809-3817] site specifically photoaffinity label protein L23 to the highest extent of any Escherichia coli ribosomal protein. In this work we demonstrate that L23 that has been photoaffinity labeled within a 70S ribosome by puromycin (puromycin-L23) can be separated from unmodified L23 by reverse-phase high-performance liquid chromatography (RP-HPLC) and further that puromycin-L23 can reconstitute into 50S subunits when added in place of unmodified L23 to a reconstitution mixture containing the other 50S components in unmodified form. We have achieved a maximum incorporation of 0.5 puromycin-L23 per reconstituted 50S subunit. As compared with reconstituted 50S subunits either containing unmodified L23 or lacking L23, reconstituted 50S subunits containing 0.4-0.5 puromycin-L23 retain virtually all (albeit low) peptidyl transferase activity but only 50-60% of mRNA-dependent tRNA binding stimulation activity. We conclude that although L23 is not directly at the peptidyl transferase center, it is sufficiently close that puromycin-L23 can interfere with tRNA binding. This conclusion is consistent with a number of other experiments placing L23 close to the peptidyl transferase center but is difficult to reconcile with immunoelectron microscopy results placing L23 near the base of the 50S subunit on the side facing away from the 30S subunit [Hackl, W., & Stöffler-Meilicke, M. (1988) Eur. J. Biochem. 174, 431-435].

Protein estimation by the product of integrated peak area and flow rate.

A convenient method for protein estimation is described, making use of uv detectors and peak integrators that are standard equipment on modern high-performance liquid chromatographs to determine the product of integrated peak area and flow rate of eluting protein at 214 nm (AF214). We demonstrate that AF214 is proportional to the amount of eluted protein and describe two approaches for calibrating the integrator, by quantitative amino acid analysis and by determining the elution yield of a known amount of applied protein, allowing direct estimation of protein from AF214. Both approaches yield similar results. The basis for the method is that, for virtually all proteins, absorbance at 214 nm is dominated by the summed contributions from the peptide groups. More accurate estimates can be made when the amino acid composition of the eluting protein is known, since this permits a correction to be made for contributions of amino acid side chains to absorbance at 214 nm. Comparison of AF214 estimates for proteins from the small (30 S) subunit of the Escherichia coli ribosome with those obtained by Bradford analysis shows the latter to give somewhat higher values.

In vitro methylation of Escherichia coli 16S ribosomal RNA and 30S ribosomes.

Treatment of synthetic 30S particles lacking all of the normally methylated nucleotides with S-adenosyl-[3H]methionine and either an S100 or ribosomal high salt wash extract resulted in ribosome-dependent incorporation of [3H]methyl groups into trichloroacetic acid-insoluble material. No incorporation was observed when naturally methylated isolated 30S particles were used, showing that methylation at unnatural sites did not occur. Enzymatic hydrolysis of the labeled RNA to nucleosides followed by HPLC analysis identified the [3H]methylated residues. Activities for the formation of N6-methyladenosine, N6-dimethyladenosine, 5-methylcytidine (m5C), 3-methyluridine, and N2-methylguanosine were found. Fractionation by ammonium sulfate partially resolved the different activities. All of the fractions with m5C activity were 6-8 times more active on synthetic unmethylated 16S RNA than on synthetic 30S ribosomes, whereas the N2-methylguanosine activity preferred 30S ribosomes to 16S RNA by a factor of more than 10. The N6-methyladenosine and N6-dimethyladenosine activities were 30S ribosome-specific. The m5C activity present in the 55-85% ammonium sulfate fraction of the high salt wash yielded a maximum of 1.0 mol of m5C per mol of 16S RNA, although two m5C residues, positions 967 and 1407, are found in vivo. RNase protection by hybridization with the appropriate oligodeoxynucleotide identified the methylated residue as C-967. Methylation of m5C-967 did not require prior methylation of G-966, and methylation of A-1518 and A-1519 was not dependent on prior methylation of G-1516.

In vitro assembly of 30S and 70S bacterial ribosomes from 16S RNA containing single base substitutions, insertions, and deletions around the decoding site (C1400).

An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes [Krzyzosiak et al. (1987) Biochemistry 26, 2353-2364] was used to make 10 single base changes around C1400, a residue known to be at the decoding site. C1400 was replaced by U, A, or G, five single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400. Another mutant possessed seven additional nucleotides at the 3' end of the 16S RNA such that a stem and loop involving the anti-Shine-Dalgarno sequence could form. Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomes. Modified in vitro reconstitution conditions were required to obtain assembly of all of the synthetic ribosomes. Quantitative HPLC analysis of the protein content of each mutant showed that all of the proteins were present. The ability of synthetic 30S to form 70S particles under functional assay conditions was about 75% that of natural 30S and was unchanged by any of the mutations except for the deletion of G1401, which decreased the association activity under the standard conditions to 35-40% of synthetic 30S. That part of the ribosomal P site which interacts with the anticodon loop of tRNA was investigated by near-UV (greater than 300 nm) induced cross-linking of AcVal-tRNA. Cross-linking depended on both 30S subunits and the correct codon. The cross-linking yield of all mutants with a pyrimidine at position 1400 was equal to control isolated 30S, and the first-order rate constants for cross-linking of those mutants tested were like reconstituted natural 30S. The site of cross-linking for mutants with a C or U insertion between C1400 and G1401 was shifted to the inserted residue. Cross-linking to the base 5' to G1401 rather than to the residue 3' to C1399 indicates that G1401 is an important structural determinant of the P site.

Effect of point mutations in the decoding site (C1400) region of 16S ribosomal RNA on the ability of ribosomes to carry out individual steps of protein synthesis.

In order to probe the relationship between structure and function of the ribosome, an in vitro system [Denman et al. (1988) Biochemistry (preceding paper in this issue)] was used to make a series of base changes around C1400, a residue known to be at the decoding site. Replacement of C1400 by U, A, or G, deletion of single bases at and to either side of C1400, and insertion of C or U next to C1400 were done. In a separate study, a mutant with seven extra nucleotides at the 3' end was constructed. The activity of these 11 mutants in A and P site binding and in initiation-dependent and initiation-independent peptide synthesis was analyzed. None of the base substitutions of C1400 were markedly inhibitory despite the almost complete conservation of this residue in ribosomal RNAs from a wide range of species. The insertions and deletions completely blocked initiation-dependent peptide synthesis but markedly stimulated the initiation-independent reaction. The effects on tRNA binding were variable. The extra stem and loop at the 3' end blocked initiation-dependent peptide synthesis but did not influence the other assays. The only modification to block all ribosomal function was the deletion of G1401. It appears that while the conserved and cross-linkable C1400 is not essential for function, the adjacent conserved G1401 is.

The role of 16S RNA in ribosome function: single base alterations and their effect on in vitro protein synthesis.

An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes (Krzyzosiak et al., Biochemistry 26, 2353-2364, 1987) was used to make 10 single base changes around C1400, the residue known to be at the decoding site. C1400 was replaced by U, A, or G, 5 single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400. Another mutant possessed 7 additional nucleotides at the 3' end of the 16S RNA such that a stem and loop involving the anti-Shine-Dalgarno sequence could form. Another series of 8 mutants tested hypothetical base-pairing between C1404, G1405 and C1496, G1497. The activity of these 19 mutants in A and P site binding, and in initiation-dependent and initiation-independent peptide synthesis was determined. None of the base substitutions of C1400 were strongly inhibitory. The insertions and deletions completely blocked initiation-dependent peptide synthesis but markedly stimulated the initiation-independent reaction. The effects of tRNA binding were variable. The only alteration to block all ribosomal function was the deletion of G1401. The extra stem and loop at the 3'-end blocked initiation-dependent peptide synthesis, but all other assays were normal. The mutants which break and reform the hypothetical base-pairs had a functional pattern that suggest the contiguous base-pairs do exist and are functionally important.

On the structural specificity of puromycin binding to Escherichia coli ribosomes.

We have examined the structural specificity of the puromycin binding sites on the Escherichia coli ribosome that we have previously identified [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry 19, 3809-3817, and references cited therein] by examining the interactions of a series of adenine-containing compounds with these sites. We have used as measures of such interactions the inhibition of [3H]puromycin photoincorporation into ribosomal proteins from these sites, the site-specific photoincorporation of the 3H-labeled compounds themselves, and the inhibition of peptidyl transferase activity. For the first two of these measures we have made extensive use of a recently developed high-performance liquid chromatography (HPLC) method for ribosomal protein separation [Kerlavage, A. R., Weitzmann, C., Hasan, T., & Cooperman, B.S. (1983) J. Chromatogr. 266, 225-237]. We find that puromycin aminonucleoside (PANS) contains all of the structural elements necessary for specific binding to the three major puromycin binding sites, those of higher affinity leading to photoincorporation into L23 and S14 and that of lower affinity leading to photoincorporation into S7. Although tight binding to the L23 and S7 sites requires both the N6,N6-dimethyl and 3'-amino groups within PANS, only the N6,N6-dimethyl group and not the 3'-amino group is required for binding to the S14 site. Our current results reinforce our previous conclusion that photoincorporation into L23 takes place from the A' site within the peptidyl transferase center and lead us to speculate that the S14 site might be specific for the binding of modified nucleosides. They also force the conclusion that puromycin photoincorporation proceeds through its adenosyl moiety.

Application of high-performance liquid chromatography to the reconstitution of ribosomal subunits.

We are currently utilizing reversed-phase high-performance liquid chromatography (RP-HPLC) in reconstitution experiments designed to study the structure and function of Escherichia coli ribosomes. The applications of RP-HPLC in these experiments include: (a) preparation of individual proteins or groups of proteins on a milligram scale for reconstitution pools, (b) analysis of the protein stoichiometry of reconstituted subunits, (c) determination of the extent and specificity of modification of proteins extracted from ribosomal subunits which have been subjected to chemical modification, and (d) resolution of modified forms of proteins S14 and L23 from the corresponding unmodified proteins. Proteins prepared by RP-HPLC from 30S and 50S ribosomal subunits were found to reconstitute into 30S and 50S subunits respectively, as well as into slower sedimenting particles. The reconstituted subunits contain a full complement of proteins and are active in ribosomal function assays, whereas the slower sedimenting particles lack several proteins and have little or no activity.

Reversed-phase high-performance liquid chromatography of Escherichia coli ribosomal proteins. Characteristics of the separation of a complex protein mixture.

We have previously reported the application of reversed-phase high-performance liquid chromatography (RP-HPLC) to the separation of Escherichia coli ribosomal proteins (A. R. Kerlavage, L. Kahan and B. S. Cooperman, Anal. Biochem., 123 (1982) 342-348; A. R. Kerlavage, T. Hasan and B. S. Cooperman, J. Biol. Chem., in press). In the present studies RP-HPLC is shown to yield much greater resolution of these proteins than does size-exclusion HPLC. In addition, we report on various aspects of RP-HPLC of ribosomal proteins including column capacity, resolution, reproducibility, recovery, separation of irreversibly denatured protein, and analysis of affinity-labeled ribosomal protein. The capacity of analytical columns was found to range from several micrograms to several milligrams with minimal loss in resolution and highly reproducible retention values. Recovery varied from protein to protein and ranged from 27% to 91%, with an average total protein recovery of 70%. The partitioning of several proteins between two peaks was shown to be due to irreversible denaturation of a small fraction. Finally, the utility of RP-HPLC in the study of the ribosome was demonstrated by analyses of [3H]puromycin-labeled ribosomal proteins, and the demonstration that labeling slightly alters protein elution.