Base-Pairing between 23S rRNA and tRNA in the Ribosomal A Site

Molecular Cell, Vol. 4, 859–864, November, 1999, Copyright 1999 by Cell Press

Base-Pairing between 23S rRNA and tRNA in the Ribosomal A Site Daniel F. Kim and Rachel Green* Department of Molecular Biology and Genetics Johns Hopkins University School of Medicine Baltimore, Maryland 21205

Summary The aminoacyl (A site) tRNA analog 4-thio-dT-p-C-ppuromycin (s4TCPm) photochemically cross-links with high efficiency and specificity to G2553 of 23S rRNA and is peptidyl transferase reactive in its cross-linked state, establishing proximity between the highly conserved 2555 loop in domain V of 23S rRNA and the universally conserved CCA end of tRNA. To test for base-pairing interactions between 23S rRNA and aminoacyl tRNA, site-directed mutations were made at the universally conserved nucleotides U2552 and G2553 of 23S rRNA in both E. coli and B. stearothermophilus ribosomal RNA and incorporated into ribosomes. Mutations at G2553 resulted in dominant growth defects in E. coli and in decreased levels of peptidyl transferase activity in vitro. Genetic analysis in vitro of U2552 and G2553 mutant ribosomes and CCA end mutant tRNA substrates identified a base-pairing interaction between C75 of aminoacyl tRNA and G2553 of 23S rRNA. Introduction The ribosome is the two-subunit ribonucleoprotein particle responsible for translation of the genetic material into the encoded polypeptides. Two-thirds of the mass of the ribosome is composed of RNA (5S, 16S, and 23S in bacteria), and considerable biochemical and genetic evidence indicates that the rRNAs are fundamentally involved in the various functions of the ribosome including decoding, translocation, tRNA binding, and peptidyl transferase (PT) (reviewed in Lieberman and Dahlberg, 1995; Green and Noller, 1997). During translation, peptide bond formation occurs between tRNA substrates bound in the aminoacyl (A) and peptidyl (P) sites of the ribosome. An important step in understanding the coordinated events in translation is the definition of the molecular interactions involved in orienting these tRNA substrates for catalysis. The CCA end of the tRNA substrates of the ribosome is universally conserved and is known to be involved in critical interactions with the ribosome. Chemical modification and cross-linking approaches have identified in domains IV and V of 23S rRNA highly conserved and accessible nucleotides that are proximal to the tRNA substrates (reviewed in Kirillov et al., 1997). Efficient cross-linking between a benzophenone derivatized Phe-tRNAPhe and several universally conserved nucleotides in the central loop of domain V indicates its proximity to the amino * To whom correspondence should be addressed (e-mail: ragreen@ jhmi.edu).

acid moieties of the A and P site tRNAs (Steiner et al., 1988). Footprinting experiments identified a small number of nucleotides in 23S rRNA that were specifically protected by P site–bound tRNA (Moazed and Noller, 1989). One of these nucleotide protections led to the identification of a Watson–Crick base-pairing interaction between G2252 in domain V of 23S rRNA (a universally conserved nucleotide) (Samaha et al., 1995) and C74 of P site bound tRNA. Interactions between other conserved nucleotides of domain V and the universal A76 and C75 of peptidyl tRNA remain elusive (Samaha et al., 1995; Spahn et al., 1996b; Green et al., 1997; Saarma et al., 1998) although a Hoogsteen pairing interaction between U2585 and A76 has been proposed (Porse et al., 1996). In the A site, tRNA footprinting analysis similarly identified nucleotides in domain V of 23S rRNA that were protected by A site–bound tRNA and that might therefore be involved in direct rRNA–tRNA interactions; these positions included both G2553 and U2555 in the highly conserved 2555 loop (Moazed and Noller, 1989). Recently, we identified a highly specific and efficient crosslink between a photoactivated aminoacyl tRNA analog, 4-thio-dT-p-C-p-Puromycin (s4TCPm), and G2553 of 23S rRNA (Green et al., 1998); this cross-linked s4TCPm substrate is highly reactive in the peptidyl transferase reaction. These data place the CCA end of A site tRNA in the vicinity of the 2555 loop of domain V of 23S rRNA (Figure 1). Two additional experiments place the 2555 loop near the CCA end of the A site tRNA: genetic studies identified a ribosomal frameshift suppressor mutation at position U2555 that was proposed to function by altering the recognition of A site–bound tRNAs (O’Connor and Dahlberg, 1993) and modification interference analysis identified U2555 as critical for a functional interaction with puromycin (Bocchetta et al., 1998). The antibiotic puromycin functions as a minimal A site substrate though its affinity for the ribosome is not extremely high (Kd 100–400 mM) (Odom and Hardesty, 1992). Early studies indicated that the addition of both C74 and C75 to this minimal substrate resulted in substantial increases in binding affinity and acceptor activity in vitro (Chladek, 1980; Bhuta et al., 1982). Subsequently, mutational analysis of positions C74 and C75 was performed; mutations at C74 had little effect on acceptor binding affinity or function while mutations at C75 resulted in 2- to 20-fold effects on these same properties (Tezuka and Chladek, 1990). We have observed similar contributions of C74 and C75 to interaction with the ribosomal A site using synthetic puromycin derivatives (data not shown). In this study, site-directed mutations were made at the universally conserved positions U2552 and G2553 (E. coli numbering) of 23S rRNA and studied both in vivo in E. coli and in our recently described in vitro reconstitution system for B. stearothermophilus (Green and Noller, 1999). In vitro genetic analysis with minimal A site tRNA analogs derived from puromycin led to the conclusion that G2553 of 23S rRNA base-pairs with C75 of A site tRNA. These data, taken together with the previously identified P site interaction (Samaha et al.,

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Figure 1. Secondary Structures Showing the Specific Nucleotides and the Location of the 2555 Loop of 23S rRNA (A) The 2555 loop (A loop) found in domain V of E. coli 23S rRNA. Universally conserved nucleotides are indicated with a gray square, the nucleotides protected from chemical modification by A site tRNA are circled (Moazed and Noller, 1989), and the site of cross-linking (2553) by 4-thio-dT-p-C-p-puromycin (s4TCPm) (Green et al., 1998) is indicated with an arrow. Nucleotide positions Um2552 and G2553 are indicated. (B) Schematic of domain V of E. coli 23S rRNA showing the locations of the 2250 and 2555 loop regions implicated in direct pairing interactions with the P site (Samaha et al., 1995) and A site tRNA, respectively (the P and A loops).

1995), implicate 23S rRNA in direct binding interactions with both the substrates of peptidyl transferase, emphasizing the fundamental role played by the 23S rRNA in the function of the ribosome. Results and Discussion In this study, we describe the identification of a pairing interaction between C75 of aminoacyl tRNA and G2553 in the highly conserved 2555 loop of domain V of 23S rRNA (A loop). This loop had previously been identified as A site proximal by a number of different approaches (see Introduction). The primary sequence of the 2555 loop of 23S rRNA is highly conserved. U2552, G2553, and C2556 are universally conserved nucleotides, whereas U2554 and U2555 are highly conserved with an occasional C substitution in the archaeal lineage (Gutell et al., 1992) (Figure 1A). In Vivo Analysis of Site-Directed Mutations at Um2552 and G2553 To investigate the possibility of base-pairing between the 2555 loop of 23S rRNA and A site tRNA, mutations were introduced at positions Um2552 and G2553 of E. coli 23S rRNA genes as part of an rrnB operon-containing plasmid construct under transcriptional control of the inducible lambda PL promoter (Samaha et al., 1995). Expression of the mutant E. coli 23S rRNA is induced by growth at 428C. The mutant 23S rRNA molecules carry the G2058 mutation conferring erythromycin resistance, whereas chromosomally encoded 23S rRNA are sensitive to erythromycin. Thus, dominant phenotypes of plasmid-encoded rRNA mutants are examined on media containing ampicillin to select for the expression plasmid, and recessive phenotypes are analyzed on media also containing erythromycin. Expression of 23S rRNAs containing mutations (A, C, or G) at Um2552 resulted in no dominant growth defect (Figure 2A); these data are consistent with earlier experiments by Porse and Garrett (1995). Mutations G2553A and G2553U had severe dominant growth defects and G2553C had a mild dominant growth defect (Figure 2A). Under these growth

conditions, all mutant rRNAs were expressed and incorporated into 70S ribosomes, though several mutant rRNAs (Um2552A, G2553A, and G2553U) were underrepresented (by ca. 2-fold) in the active polysome fraction (data not shown). When these same mutant rRNAs were grown on erythromycin-containing media, all six mutations result in severe growth defects, though there was some residual growth for the Um2552 mutant 23S rRNAs, especially Um2552C (data not shown). There are numerous examples where mutation of universally conserved nucleotides in 23S rRNA yielded no dominant growth defect (Lieberman and Dahlberg, 1994; Porse and Garrett, 1995; Spahn et al., 1996a; Green et al., 1997). These data are consistent with a critical role for G2553 in the function of the ribosome. Analysis of U2552 and G2553 Mutant 23S rRNAs in Peptidyl Transferase Reactions Next, we looked at the peptidyl transferase activity of U2552 and G2553 mutant ribosomes in vitro. Mutant 50S ribosomal subunits were reconstituted from in vitro– transcribed B. stearothermophilus 23S rRNA according to (Green and Noller, 1999). The peptidyl transferase activity of these ribosomes was assayed using the “fragment reaction” with the minimal peptidyl and aminoacyl tRNA substrates, CCACCA-N-Ac-[35S]-Met and puromycin, respectively (Monro and Marcker, 1967); the products of the reaction were resolved by paper electrophoresis (Green and Noller, 1996). None of the mutations at U2552 resulted in significant decreases in peptidyl transferase activity. Porse and Garrett previously reported 2- to 4-fold effects for the Um2552A and Um2552C mutations in E. coli ribosomes using the same assay (Porse and Garrett, 1995). At G2553, only the G2553C mutant ribosomes had substantially (10-fold) reduced PT activity in the fragment reaction (Figure 2B). To further characterize the mutant reconstituted 50S subunits, a peptidyl transferase assay was developed where the A site substrate was provided in limiting amounts relative to the ribosome concentration (and relative to its binding affinity, Km, for the ribosome); here, substrate binding defects can be detected as decreases

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Figure 3. Suppression Analysis of G2553 Mutant Ribosomes with C74 and C75 tRNA Mutant A Site Substrates Figure 2. Effect of Mutations at U2552 and G2553 of 23S rRNA on Growth and Peptidyl Transferase Activity (A) Expression of rRNA genes carrying mutations at positions Um2552 and G2553 of 23S rRNA. DH1 E. coli cells transformed with the wild-type plasmid, pLK45, or the indicated mutant versions were plated on solid medium containing Amp40-Kan50 at 428C. (B) Fragment peptidyl transferase assay. Phosphorimager exposure of paper electrophoresis analysis of PT “fragment” reaction catalyzed by wild-type and U2552 and G2553 mutant versions of 23S rRNA incorporated into B. stearothermophilus 50S subunits. Spots represent the product N-Ac-[35S]-Met-puromycin. (C) A-site limiting peptidyl transferase assay. Phosphorimager exposure of polyacrylamide denaturing gel resolving the input 59-[32P]-labeled CPm derivative from the product of the PT reaction ([32P]-CPm-N-Ac-Phe) catalyzed by U2552 and G2553 mutant B. stearothermophilus 50S subunits reconstituted from in vitro–transcribed 23S rRNA.

in the rate of the PT reaction. In the standard “fragment reaction,” aminoacyl substrate (puromycin) is present at a saturating concentration (1 mM) relative to its Km. Reconstituted B. stearothermophilus 50S subunits (Green and Noller, 1999) were combined with highly purified E. coli 30S subunits to form 70S ribosomes (Nomura et al., 1968; Fahnestock et al., 1974). Next, the peptidyl (P) site was filled with N-Ac-Phe-tRNAPhe using poly(U) as an mRNA template. The PT reaction was initiated with synthetic A site substrates (59-[32P]-labeled puromycin derivatives [NPm and NCPm, where N 5 A, C, G or U]). After the PT reaction, labeled starting material ([32P]NNPm) was resolved from the product of the PT reaction ([32P]-NNPm-Phe-Ac-N) on polyacrylamide denaturing gels. 50S subunits reconstituted from in vitro–transcribed 23S rRNA were ca. 15-fold less active than natural B. stearothermophilus 50S subunits; however, this level of activity was readily detected and quantitated (data not shown). Next, we examined the effects of mutations at U2552

(A) Wild-type and mutant reconstituted B. stearothermophilus 50S subunits were incubated with 59-[32P]-labeled C74 mutant puromycin derivatives of the form NCPm (where N 5 C, A, G, or U, respectively) in the A site limiting assay. Quantitated data were expressed on the y axis as percentage of input substrate converted to product. The Watson–Crick pairing partner is shaded dark gray in each set of four to highlight predicted patterns. (B) As in (A) except with 59-[32P]-labeled C75 mutant puromycin derivatives of the form NPm (where N 5 C, A, G, or U, respectively). In both (A) and (B), standard deviations are included from a compilation of four experiments.

and G2553 using this A site assay. Mutations at U2552 still had less than a 2-fold effect on PT activity, even with limiting A site substrate (Figure 2C). However, all mutations at G2553 resulted in at least 10-fold decreases in activity (Figure 2C) with limiting [32P]-CPm substrate. These data suggest that G2553 of 23S rRNA is involved in interactions important for A site function. The more severe effects on PT activity of the G2553 mutations in the alternative A site assay can be explained (1) by the use of limiting substrate concentrations where binding differences are more readily detected and (2) by the use of minimal A site substrates (CPm) that maximize the relative contribution of the CCA end of the tRNA to binding. Analogously, the P site Watson–Crick interaction was identified in an assay using limiting concentrations of minimal P site substrates. Indeed, when intact tRNA substrates were used, binding deficiencies at the CCA end were masked by strong interactions of the anticodon end of tRNA with the 30S subunit of the ribosome (Samaha et al., 1995). Compensatory Analysis of G2553 Mutant Ribosomes and Mutant Aminoacyl tRNA Substrates (NNPm) Given the decreased PT activity of the G2553 mutant ribosomes and the established proximity between the

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CCA end of aminoacyl tRNA and G2553 of 23S rRNA, we next asked whether mutations at C74 or C75 of aminoacyl tRNA could suppress the deleterious in vitro phenotype of these mutations. When assayed with the NCPm A site substrates, where N is any of the four nucleotides (A, C, G, or U), each of the G2553 mutants had diminished overall activity (Figure 3A); these data are consistent with the results observed with the CPm substrate (Figure 2C). Each of the NCPm substrates had differing activities (for example, up to 3-fold differences were observed between the CCPm or GCPm substrates and the UCPm substrate), but the relative activities of the four NCPm substrates were similar regardless of the mutant ribosome used. Specificity conferred by the C74 nucleotide was independent of the identity of the nucleotide at G2553 of the ribosome. These subtle differences in activity associated with C74 mutations in the A site are consistent with previous experiments (Tezuka and Chladek, 1990; O’Connor et al., 1993). The pattern of reactivity observed was not consistent with the existence of an interaction between C74 and G2553 (Figure 3A). When the same experiment was performed with NPm substrates, where N again is any of the four nucleotides, a distinct pattern of suppression emerged (Figure 3B). The wild-type ribosomes, G2553, preferred the wild-type substrate (CPm) by about 2- to 5-fold relative to the other substrates (APm, GPm, and UPm). Furthermore, this substrate that was preferred by wild-type G2553 ribosomes (CPm) was least active with all of the mutant ribosomes (G2553A, G2553C, and G2553U), emphasizing that the pairing specificity has been changed by these mutations. In the clearest example of suppression, the G2553C ribosomes preferred the GPm substrate by at least 20-fold relative to the other three substrates, demonstrating a direct and specific interaction between C75 of A site tRNA and G2553 of the ribosome. Indeed, with the G2553C ribosome, activity was restored to levels higher than those observed with the wild-type ribosome and substrate. These data are consistent with and suggestive of a simple Watson–Crick pairing interaction; however, other models, including, for example, reverse Watson–Crick pairing, could also be accomodated by these results. In the case of the G2553U mutant ribosomes, activity was low with the CPm substrate, but substantially (6-fold) enhanced with the APm substrate, and somewhat less enhanced (2- to 3-fold) with the GPm and UPm substrates. For the G2553A mutant ribosomes, like the G2553U mutant ribosomes, PT activity was low with the CPm substrate. Substantially (7-fold) enhanced activity was observed with the GPm substrate, and somewhat less enhanced activity (3- to 4-fold) with the APm and UPm substrates. Thus, only in the case of the G2553A ribosomes is the canonical Watson–Crick pairing partner not the preferred substrate. Indeed, the activity of the GPm substrate with the G2553A ribosomes helps us to distinguish between the possibilities of normal and reverse Watson–Crick pairing interactions since a G:A base pair can not readily be modeled in a reverse configuration. A two-hydrogen bond interaction between G and A is, however, stable and well accomodated into helices in stacked intrahelical forms (Dodgson and Wells, 1977a,

Figure 4. Nucleotide Analog 2,6-diaminopurine Tests Watson–Crick Face Pairing Interaction (A) Chemical structures of Watson–Crick-type base pairs between uridine and adenosine (U:A) and between uridine and 2,6-diaminopurine (U:D) with two and three hydrogen bonds, respectively. (B) Deoxyadenosine-puromycin (dA-Pm) and deoxy-2,6-diaminopurine-puromycin (d26DAP-Pm) substrates were compared in the A site limiting assay for their level of activity on G2553 and G2553U mutant ribosomes. Quantitated data were expressed on the y axis as the percentage of input substrate converted to product. Standard deviations are included from a compilation of four experiments.

1977b). NMR studies of double-stranded DNA dodecamers with two G:A pairs surrounded by canonical Watson–Crick pairing found low exchange of the imino protons of the guanosines involved in the G:A pairs at low temperature, indicative of a stable helical configuration (Patel et al., 1984). G:A pairs are included in the secondary structure of the rRNAs as confirmed by covariation analysis (Noller and Woese, 1981; Noller et al., 1981). In one study of template directed fidelity at ligation sites, G:A pairs were actually favored over Watson–Crick pairing partners at low temperature (James and Ellington, 1997). The high peptidyl transferase activity observed with the G2553A mutant ribosome and the GPm substrate is most consistent with a normal Watson–Crick face pairing where a stable two-hydrogen bond interaction occurs. 2,6-Diaminopurine Analog Supports Model for Pairing Interaction between G2553 and C75 of A Site tRNA The suppression patterns observed with the four standard ribonucleotides were consistent with a normal Watson–Crick pairing interaction between C75 of A site

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tRNA and G2553 of 23S rRNA. As a further test of whether the interaction between these two nucleotides was taking place on the Watson–Crick face of the base moiety, a 2,6-diaminopurine deoxynucleotide analog was incorporated into a dinucleotide puromycin derivative (NPm). Because 2,6-diaminopurine has an additional amino function at position 2 of the purine ring, an extra hydrogen bond can form with uridine, forming a more stable base pair with three hydrogen bonds (Figure 4A). The activity of this substrate (d26DAP-Pm) was compared with that of an equivalent deoxynucleotide adenosine substrate (dA-Pm) on the panel of G2553 mutant ribosomes. The activity of the G2553U ribosome increased by ca. 10-fold with the 2,6-diaminopurine substrate compared with the adenosine substrate. By contrast, the activity of the wild-type G2553 ribosomes was only moderately (2-fold) increased with the same 26DAP-Pm substrate (Figure 4B). Further analysis of nucleotide analogs might reveal other aspects of this rRNA–tRNA interaction. Conclusions The results presented identify the existence of a direct interaction between C75 of A site tRNA and G2553 of domain V of 23S rRNA. The data are most consistent with a Watson–Crick-type interaction. In the previous identification of a direct interaction between G2252 of 23S rRNA and C74 of peptidyl tRNA, the pattern of suppression was unambiguously indicative of a Watson– Crick pairing interaction (Samaha et al., 1995). Here, while the wild-type and G2553C ribosomes were highly specific for one (Watson–Crick) substrate, the G2553A and G2553U ribosomes were less specific. This might be explained as follows. The G2553 and G2553C ribosomes may fold into well constrained three-dimensional active site structures where A site substrate discrimination is maintained. The decreased PT activity of the G2553C mutant ribosomes in the fragment assay, where no C75 equivalent nucleotide is present, might be explained by a conformationally distorted 2555 loop in the G2553C ribosomes where the conformation is only restored by an appropriate pairing interaction with the A site tRNA. On the other hand, the G2553A and G2553U mutant ribosomes might be less conformationally constrained and more promiscuous in their interaction with the A site substrate. Such promiscuous pairing interactions apparently have been exploited by the U5 snRNP to accomodate the heterogeneous exonic splice sites found in the genome (Newman and Norman, 1992; Sontheimer and Steitz, 1993). Structural studies of the ribosome may provide a molecular explanation for our observed differences in specificity. For example, NMR studies of the 2250 loop of 23S rRNA, the site of interaction of C74 of peptidyl tRNA, have provided a molecular rationale for the ability of G2252 mutant ribosomes to interact efficiently with mutant tRNA substrates: changes in the structurally isolated G2252 are unlikely to result in global changes in the conformation of the P loop (Puglisi et al., 1997). Both the aminoacyl and peptidyl tRNA substrates of the ribosome are directed to the catalytic core via direct RNA–RNA interactions with 23S rRNA (Figure 1B). As the remainder of the catalytic core of the ribosome is revealed by functional and structural (Ban et al., 1999;

Cate et al., 1999) studies, it will be interesting to see whether different regions of the rRNA actively participate in the chemical steps of catalysis. Experimental Procedures Site-Directed Mutations in E. coli and B. stearothermophilus 23S rRNA Oligonucleotide-directed mutations were constructed in pBS23S (E. coli 23S rRNA) as described (Samaha et al., 1995). Expression plasmids in pLK45 were constructed as described (Samaha et al., 1995). Oligonucleotide-directed mutations were constructed in pBST7–23S (B. stearothermophilus 23S rRNA) as described (Green and Noller, 1999). Mutations at U2552 in B. stearothermophilus were constructed by cassette mutagenesis into pBST7–23S following amplification by the polymerase chain reaction (PCR) of the region of interest with oligonucleotide primers containing the PflM1 and HindIII restriction sites. In Vivo Analysis of 23S rRNAs with Mutations at Um2552 and G2553 Mutant E. coli pLK45 derivative plasmids were transformed into E. coli strain DH1 containing plasmid pcI857, and the in vivo analysis was performed as previously described (Green et al., 1997). In Vitro Reconstitution of B. stearothermophilus 50S Subunits B. stearothermophilus 50S subunits were reconstituted from in vitro transcripts (wild-type and mutant versions) of 23S rRNA as previously described (Green and Noller, 1999). Fragment Reaction—A-Site Saturating Assay N-protected aminoacylated initiator methionine tRNA fragment (CAACCA-N-Ac-[35S]-methionine) was prepared and peptidyl transferase activity was measured as described (Green and Noller, 1996). Each 100 ml reaction contained 12.4 pmol of 50S subunits (or 30 ml of a standard reconstitution reaction) and 2.5 pmol CCACCA-N-Ac[35S]-methionine in 0.4 M KOAc, 50 mM Tris-HCl (pH 8.3) and 60 mM MgCl2. Reactions were initiated with puromycin (1 mM final in aqueous) and 50 ml of methanol. Products of the reaction were resolved by paper electrophoresis and quantitated as previously described (Green and Noller, 1996). Synthesis and Purification of Puromycin Derivatives Oligonucleotide derivatives of the aminoacyl substrate puromycin were chemically synthesized by solid-phase phosphoramidite chemistry. CPG-puromycin was originally synthesized as described (Green et al., 1998) and eventually was obtained commercially from Glen Research. Deoxynucleotide puromycin derivatives (dAPm and d26DAP-Pm) and ribonucleotide puromycin derivatives (NPm and NCPm) were synthesized and deprotected following the published protocols of Glen Research. All oligonucleotides were purified by reverse-phase HPLC on a C18 column (Rainin Microsorb Short-One) in buffer A (20 mM NH4OAC [pH 7.0]/10% acetonitrile) and elution with 100% acetonitrile. The identity of the di- and trinucleotide products was confirmed by negative ion electrospray mass spectrometry. A-Site Substrate Limiting Assay Reconstituted 50S subunits from B. stearothermophilus (3.4 pmol of 23S rRNA) were incubated with 3.4 pmol highly purified E. coli 30S subunits (prepared essentially as described in Merryman et al., 1999a, 1999b) and 5 mg poly(U) and 10 pmol N-Ac-[14C]-Phe-tRNAPhe at 458C for 60 min; limiting amounts (2 pmol) of radioactively labeled puromycin derivatives (NNPm kinased with g-[32P]-ATP and T4 polynucleotide kinase) were added to start the peptidyl transferase reaction. Time points were taken by stopping the reaction with an equal volume urea loading dye (7 M urea/0.13 TBE/0.1% bromphenol blue/0.1% xylene cyanol). Samples were loaded on 24%/6 M urea/ 13 TBE acrylamide gels to resolve the radioactively labeled substrate species from the more slowly migrating puromycin-phenylalanine product. Radioactive species were quantitated using the Molecular Dynamics Phosphorimager system.

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Acknowledgments

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