Crossunking of Tbe Anticodon of P Site Bound Trna to C-1400 of E.coli 16s Rna Does Not Require the Participation of Tbe 50s Subunit

CrossUnking of the 5'-ant1codon base of ribosomal P s i te bound AcVal-tRNA to residue C-1400 of 16S RNA or to I ts equivalent 1n 18S RNA has been shown to occur on 70S or 80S ribosomes of both prokaryotes and eukaryotes [C1es1olka, J . , Nurse, K., K l e i n , J . and Ofengand, J. (1985) Biochemistry 24, 3233-3239]. In the present work, we show that the cross!Inking rate, crossiinking y i e l d , and s i t e of c ross l i nk ing are a l l unchanged when the 50S subunit 1s omit ted. Therefore, a l l of the posit ional features of tRNA-r1bosome complexes which allow cross l ink ing to occur are e n t i r e l y contained 1n the 30S subunit. Blockage of reverse t ranscr ipt ion by crosslink formation was used to determine the s i te of c ross l i nk ing . This analysis revealed that RNA modi f i ca t ions which do not d i r e c t l y block base-pa1r1ng Ugands sometimes al low the modified base to be t ranscr ibed, leading to doublet band formation even when there 1s only a single crosslink s i t e . INTROOUCTIOH Previous work from this laboratory has established that certain tRNAs, placed 1n the P s i te of ribosomes from both prokaryotes and eukaryotes can be crosslinked to the small ribosomal subunit by I r radiat ion with l ight of 310-330 nm (1-4). The crosslink 1s a cyclobutane dimer (5) which binds the 5'-ant1codon tRNA base, mô u o r cmô U 1n those species which can be crosslinked, to a specific C residue 1n the 16S or 18S rRNA (6-8). The residue Involved, C-1400 1n £. coif, 1s located 1n the center of a highly conserved 17-nucleotide sequence found 1n v i r t ua l l y unaltered form 1n prokaryotic, eukaryotic, and organelle ribosomes. The retention across species lines of both sequence and close proximity to the anticodon of tRNA Is strongly suggestive of an Important role for this sequence In some ribosomal process l i ke l y related to mRNA translat ion, but so far no specif ic functional or structural role has been assigned. In a l l previous studies, 70S or 80S ribosomes were used, since A and P sites are best defined on the entire ribosomal particle. However, since the crosslink 1s ent i re ly to the small ribosomal subunit, 1t was of Interest to © IRL Press Limited, Oxford, England. 1 6 5 Nucleic Acids Research determine 1f the 30S ribosome was sufficient for crosslInking. In this work we show that while the 50S subunit stimulates binding, the crosslinMng rate and yie ld, and the site of crosslinking were unchanged 1n the absence of the 50S subunit. In addition, the abil ity of reverse transcription arrest to localize cyclobutane d1mer-!1ke crosslinks was characterized. EXPERIMENTAL Materials E. coif 70S t i g h t couple ribosomes, Ac[H]Val-tRNA, and poly(U2,G) were prepared as described previously (1). pGUU and puromydn were obtained according to Ofengand and L1ou (10). 50S and 30S subunits were prepared and activated before use as described by Krzyzosiak et al. (11) except that the activation buffer contained 60 mM NH4CI Instead of 100 mM. 30S-4 and 30S-4A were prepared 1n buffer RA and stored 1n buffer Rec20. 3OS-5A and 30S-5B were separated 1n buffer RE. Storage of 30S-5A and 3OS-58 was 1n buffer Rec20 and buffer RC, respect ive ly . The buffers are described 1n Krzyzosiak et al. (11). One A250 uni t of 70S or 30S was assumed equal to 25 or 67 pmoles, respectively. The primer used for reverse transcription, complementary to residues 1431-1453, was made on an Applied Biosystems 381A synthesizer, manually cleaved, deprotected, and p u r i f i e d as described by Krzyzosiak et al. (11). [«-2p]dATP, dideoxy nucleot ides, AMV reverse transcriptase, and deoxynucleoside triphosphates were obtained as described by Nurse et al. (4). P site binding and crossHnking The react ion mixture contained 50 mM He.pes, pH 7.5, 100 mM NH4CI, 15 mM Mg(0Ac)2, 20 ug/ml polyfUg.G) or 60 yM pGUU, 150 nM Ac[H]Val-tRNA, and 136-150 nM 30S, 30S plus 50S, or 70S E. coif ribosomes except as Indicated. The 30S and 30S plus 50S reactions contained 0.4-0.7 mM OTT carried over from the ac t i va t ion reac t ion . The 70S reactions did not contain mercaptans except for the experiment of F1 g. 1. Incubation was at 37"C for 15 m1n. Irradiat ion for 25 m1n (30S) or 45 m1n (70S) was at 0°C 1n the Rayonet photoreactor with the 300 nm lamps using ca. 1 cm of Pyrex glass as a low wavelength cutoff f i l t e r (1) . Crossl1nk1ng analysis was by adsorption to membrane f i l t e r s (Schleicher and Schuell, 8A85) as described previously (1). RESULTS Site location of anticodon-crosslInked tRNA In previous reports, we have shown that AcVal-tRNA crossl Inked via Its anticodon to the 30S subunit can s t m transfer the AcVal moiety to puroraydn