Functional conservation near the 3'' end of eukaryotic small subunit RNA: photochemical crosslinking of P site-bound acetylvalyl-tRNA to 18S RNA of yeast ribosomes.

Escherichia coli acetylvalyl (AcVal)-tRNA1Val became crosslinked to both yeast and spinach chloroplast ribosomes upon irradiation (300 nm) in the presence of poly(U2,G). Yields were 25-30% and 33%, respectively, compared to 45% for E. coli. Crosslinking occurred to the P site, only to the 40S subunit, and 90% of that was to the 18S rRNA. The crosslink could be photolyzed at 254 nm with the same first-order kinetics as for the E. coli ribosome complex previously studied. The AcVal-tRNA that split off could be crosslinked again when irradiated at 300 nm, showing that the crosslink was photoreversible. There was a strong codon specificity for crosslinking. With pG-U-U, 85% crosslinking was obtained after 20 min of irradiation; with G-U-A, only 3% crosslinking occurred. All of these properties are the same as those previously reported for the E. coli ribosome crosslink that occurs via cyclobutane dimer formation between the 5' anticodon base 5'-carboxymethoxyuridine-34 and cytidine-1400 of the 16S RNA. Cytidine-1400 is in the center of a 17-mer that has been almost totally conserved among the small subunit rRNAs of all species so far examined, including yeast. Crosslinking of tRNA in the same way to both yeast and E. coli ribosomes shows that there has been a functional conservation as well in this region of the small subunit rRNA. This region may be involved in some essential aspect of the decoding process that is common to both prokaryotic and eukaryotic protein synthesis systems.     

Localization of the decoding region on the 30S Escherichia coli ribosomal subunit by affinity immunoelectron microscopy.

The decoding region of the Escherichia coli ribosome has been localized by affinity immunoelectron microscopy. Valyl-tRNA1Val, derivatized at the alpha-amino end with the dinitrophenyl group, was bound to the ribosomal P site of 70S ribosomes and crosslinked specifically to 16S RNA by 310- to 325-nm irradiation. Previous work has shown that crosslink occurs between the 5' anticodon base of the tRNA and a pyrimidine in the 3' one-third of the 16S RNA. By reaction of the dinitrophenyl group with antibody, dimers of the 30S tRNA covalent complexes were prepared containing one covalently attached tRNA per 30S subunit. Electron microscopic visualization of the antibody attached to the dinitrophenyl group located the position of the 3' end of the tRNA. Several sites, with a strong preference for the large protrusion or cleft region in the upper one-third of the subunit, were found. The multiplicity of sites is likely due to the freedom of orientation of the 3' end of the tRNA which is approximately 80 A from the site of crosslinking. By extrapolating this distance from each of the antigenic sites, we concluded that the anticodon of tRNA when in the P site is probably located in the cleft region of the 30S subunit.     

Oligonucleotides with rapid turnover of the phosphate groups occur endogenously in eukaryotic cells.

Endogenous oligonucleotides were found in trichloroacetic acid extracts of hamster lung fibroblasts and Tetrahymena cells. Peaks of radioactivity that eluted with retention times similar to oligonucleotide markers (5- to 50-mer) were found by HPLC in cells labeled briefly with 32Pi. Only minute amounts of UV-absorbing material were detected, consistent with a rapid turnover of phosphate groups. The 32P-labeled material also migrated as oligonucleotides on 20% polyacrylamide gels; it was not hydrolyzed by alkaline phosphatase but was digested by snake venom phosphodiesterase, S1 nuclease, and pancreatic RNase and was phosphorylated by T4 polynucleotide kinase. The 32P-labeled material isolated by HPLC was alkali labile and the hydrolyzate ran as nucleotides on paper chromatography. It is concluded that the oligonucleotides are mainly oligoribonucleotides, but it is possible that oligodeoxynucleotides are also present.     

Valine-Specific tRNA-like Structure in Turnip Yellow Mosaic Virus RNA

The 3' terminal nucleotide of turnip yellow mosaic virus (TYMV) RNA (23-25 S) may be esterified with valine in the presence of ATP and an enzyme preparation from Escherichia coli. The nucleotide composition near the valine-binding site is different for TYMV RNA and tRNA(Val) from cabbage, as shown by comparison of the valine adducts of nucleotides labeled with radioactive valine in T(1) RNase digests. Consequently, host tRNA(Val) is not involved in the observed charging of TYMV RNA with valine. The TYMV RNA appears to have a tRNA-like structure, at or near its 3' end, that is recognized by three different enzymes which specifically catalyze reactions involving tRNA.     

5''-Terminal nucleotide sequence of Semliki forest virus 18S defective interfering RNA is heterogeneous and different from the genomic 42S RNA

An 18S defective interfering (DI) RNA population was isolated from the cytoplasm of baby hamster kidney (BHK-21) cells infected with Semliki Forest virus from the 10th undiluted passage. The RNA was approximately 2000 nucleotides long and contained a 5'-terminal cap with the structure 7mGpppAp and a poly(A) tract. The DI RNA contained large TI oligonucleotides derived from both the 42S RNA-specific region and the 3' one-third of the genome common to 42S and 26S RNA. Several of the large oligonucleotides were present in nonequimolar ratios, suggesting that the RNA population is heterogeneous. As this population is approximately uniform in size, this suggests that the DI RNAs may be generated by internal deletions involving different regions of the genome. The 5'-terminal cap-containing RNase T1 oligonucleotide was isolated by two-dimensional gel electrophoresis from uniformly 32P-labeled RNA and shown to be heterogeneous. Five T1 caps with the structure 7mGpppA-U(A-U)nC-A-U-G(n = 4-8) were identified. The two major T1 caps (n = 4 and 6) comprised about 75% of the total yield of T1 caps. The T1 caps were different from the genomic 42S RNA T1 cap (7mGpppA-U-G), suggesting that the extreme 5' end of the genome is not conserved in this defective interfering RNA.     

How ribosomes select initiator regions in mRNA: base pair formation between the 3'' terminus of 16S rRNA and the mRNA during initiation of protein synthesis in Escherichia coli.

Initiation complexes formed by E. coli ribosomes in the presence of 32P-labeled A protein initiator region from R17 bacteriophage Rna have been treated with colicin E3 and disassembled by exposure to 1% sodium dodecyl sulfate. Electrophoresis on 9% polyacrylamide gels reveals a dissociable complex containing the 30-nucleotide-long messenger fragment and the 50-nucleotide-long colicin fragment, which arises from the 3' terminus of the 16S RNA. The complex is a pure RNA-RNA hybird; it is apparently maintained by a seven-base complementarity between the two RNA fragments. Detection of this mRNA-rRNA complex strongly supports the hypothesis that during the initiation step of protein biosynthesis the 3' end of 16S RNA base pairs with the polypurine stretch common to initiator regions in E. coli and bacteriophage mRNAs. The implications of our findings with respect to the molecular mechanism of initiation site selection and mRNA binding to ribosomes, the role of rRNA in ribosome function, and species specificity in translation are explored.     

Photo-Affinity Labeling of tRNA Binding Sites in Macromolecules. I. Linking of the Phenacyl-p-azide of 4-Thiouridine in (Escherichia coli) Valyl-tRNA to 16S RNA at the Ribosomal P Site

The phenacyl-p-azide of 4-thiouridine in (E. coli) tRNA₁^Val was prepared for use as a photo-affinity probe of tRNA binding sites on ribosomes. The derivatized tRNA was 90-100% as active as control tRNA for aminoacylation, nonenzymatic binding to the ribosomal P site, elongation factor Tu(EFTu)-dependent binding to the A site, EFTu-GTP-aa-tRNA ternary complex formation, and transfer of valine into polypeptide. Irradiation of p-azidophenacyl-[³H]valyl-tRNA bound noncovalently to the ribosomal P site resulted in covalent attachment of 15-20% of the noncovalently bound tRNA to the ribosomes. The linking occurred exclusively to the 16S RNA of the 30S ribosomal subunit, thus suggesting that the region of the ribosome within 9 Å of the 4-thiouridine of tRNA, when it is bound in the P site, is solely 16S RNA.     

Covalent linkage of a protein to a defined nucleotide sequence at the 5''-terminus of virion and replicative intermediate RNAs of poliovirus.

The 5'-terminus of poliovirus polyribosomal RNA is pUp. A candidate for the 5'-terminus of poliovirion RNA was recovered as a compound migrating toward the cathode when 32P-labeled virion RNA was completely digested with ribonucleases T1, T2 and A and analyzed by paper ionophoresis at pH 3.5. Treatment with proteinase K reversed its direction of migration, indicating the presence of protein. Treatment with venom phosphodiesterase liberated all of the radioactivity as pUp, suggesting that poliovirion RNA has a protein-pUp 5'-terminus. Treatment of virion RNA with T1 ribonuclease alone generated a proteinase K-sensitive oligoribonucleotide. Analysis of the oligoribonucleotide using ribonucleases A and U2 showed its structure to be protein-pU-U-A-A-A-A-C-A-G. Digests of replicative intermediate RNA contained sufficient protein-pUp to suggest that this structure is at the 5'-end of most nascent poliovirus RNA molecules. We suggest that a protein-nucleotide structure acts as a primer for initiating synthesis of poliovirus RNA.     

Sequence Homology at the 5′-Termini of Insect Messenger RNAs

Treatment of insect polyribosomes with 1 M KCl released a messenger ribonucleoprotein with a pronounced 16S peak. Phenol extraction resulted in a defined peak of 10S RNA, which was judged as mRNA by the following criteria: it showed specificity for binding to ribosomes, and the formation of initiation complex was dependent on protein initiation factors, GTP, mRNA, and aminoacyl-tRNA. The complex directed protein synthesis upon the addition of elongation factors. mRNA was treated with phosphatase and phosphorylated at the 5'-end with [(32)P]cyanoethylphosphate. [(32)P]mRNA was digested by T1 ribonuclease to completion and chromatographed on DEAE-cellulose. The only fragment with (32)P was 15 nucleotides long; it was treated with pancreatic ribonuclease and fingerprinted. Fractions of AC, AAC, and AAAC were found. Initiation signal AUG or GUG in these mRNAs does not begin immediately at the 5'-end and may be at a distance greater than 15 nucleotides. Alkaline hydrolysis of mRNAs labeled in vivo with [(14)C]adenosine revealed Ap and pppAp. Alkaline hydrolysis of mRNA labeled with (32)P at the 5'-terminus resulted in pAp. Hence, these results suggest that in a heterogeneous population of mRNAs from insects, all start with A and have sequence homology at the 5'-termini. This sequence may reflect the signal for RNA polymerase on the gene or may promote the binding of mRNA to ribosomes.     

Labeling the peptidyltransferase center of the Escherichia coli ribosome with photoreactive tRNA(Phe) derivatives containing azidoadenosine at the 3'' end of the acceptor arm: a model of the tRNA-ribosome complex.

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine (2N3A) at position 73 or 76 have been crosslinked to the peptidyl site of Escherichia coli ribosomes. Covalent tRNA-ribosome attachment was dependent upon the replacement of adenosine by 2N3A in the tRNA, irradiation with 300-nm light, and the presence of poly(U). In all cases, the modified tRNAs became crosslinked exclusively to 50S ribosomal subunits. While the tRNA derivative containing 2N3A at position 73 labeled only protein L27, that containing 2N3A at position 76 labeled proteins L15, L16, and L27 as well as a segment of the 23S rRNA. The site of crosslinking in the rRNA was identified as guanosine-1945, which lies within a highly conserved sequence adjacent to a number of modified bases and has not until now been identified at the peptidyltransferase center. On the basis of these results, and previously reported crosslinks from tRNA containing 8-azidoadenosine in the 3'-terminal -A-C-C-A sequence [Wower, J., Hixson, S. S. & Zimmermann, R. A. (1988) Biochemistry 27, 8114-8121], we propose a model for the arrangement of tRNA molecules at the peptidyl and aminoacyl sites that is consistent with most of the information available about the location of the peptidyltransferase center and the decoding domain of the E. coli ribosome.     

THE UTILIZATION OF GENES FOR RIBOSOMAL RNA, 5S RNA, AND TRANSFER RNA IN LIVER CELLS OF ADULT RATS

The rates of synthesis of ribosomes, 5S RNA, and tRNA necessary to maintain the steady-state concentrations of these entities in liver cytoplasm of adult rats were determined. On the average, each liver cell in the adult rat synthesizes 650 ribosomes, 650 molecules of 5S RNA, and 11,000 molecules of tRNA each minute. The numbers of genes per liver cell for rRNA, 5S RNA, and tRNA were 330, 1660, and 13,000, respectively, as determined by RNA: DNA hybridization experiments. Thus, on the average, individual genes for rRNA, tRNA, and 5S RNA are transcribed twice a minute, once a minute, and once every 2.5 minutes, respectively, in the adult rat liver.     

Site-specific cleavage by T1 RNase of U-1 RNA in u-1 ribonucleoprotein particles.

The structures and functions of small nuclear ribonucleoprotein particles have become of interest because of their suggested role in processing heterogeneous nuclear RNA [Lerner, M. R., Boyle, J. A., Mount, S. M., Wolin S. L. & Steitz, J. A. (1980) Nature (London) 283, 220-224]. To determine the conformation of U-1 RNA in U-1 ribonucleoprotein particles and whether proteins of these particles protect segments of U-1 RNA, intact particles and isolated U-1 RNA were digested with T1 RNase. The digested particles were immunoprecipitated with anti-Sm antibodies. A 5'-end fragment containing nucleotides 1-107 and 3'-end fragments containing nucleotides 108-165 and 108-153 were recovered in nearly quantitative yield from digestion of the particles, suggesting that position 107 is the principal cleavage site in them. At the same T1 RNase concentrations, deproteinized U-1 RNA was cleaved into many fragments. At low T1 RNase concentrations, major cleavage site of deproteinized U-1 RNA was at nucleotide 69. Comparison of the cleavage sites of free U-1 RNA and of U-1 RNA in U-1 ribonucleoprotein particles suggested similar secondary structures. The resistance of the 5' end of U-1 RNA to T1 RNase was unexpected inasmuch as this region has been implicated in hydrogen bonding with heterogeneous nuclear RNA splice junctions.     

Structural and Functional Properties of Ribosomes Crosslinked with Dimethylsuberimidate

To test whether a 30S ribosomal subunitformylmethionyl-tRNA-mRNA complex is an obligatory intermediate in protein synthesis, 70S ribosomes from Escherichia coli were crosslinked with the bifunctional imidoester, dimethylsuberimidate. Crosslinked ribosomes contained covelently joined 30S and 50S subunits, as judged by their inability to dissociate at low Mg(2+) concentrations. Treatment of 70S ribosomes with high salt (1 M NH(4)Cl), either before or after reaction with the crosslinking reagent, produced two different crosslinked ribosomal particles, one of "60 S" and the other "70 S." Preliminary evidence indicates that both particles can bind N-acetylphenylalanyl-tRNA at low Mg(2+) concentrations and are active for polyphenylalanine syntheses.Crosslinked ribosomes were functional when tested with poly(U) as an mRNA in systems requiring initiation factors and N-acetylphenylalanyl-tRNA for activity. Under optimal crosslinking conditions, they retained 80% of the activity of unmodified ribosomes for polyphenylalanine synthesis. Despite the maintenance of these functional capacities, such ribosomes had a sharply reduced ability to bind fMet-tRNA and were completely inactive in protein synthesis with bacteriophage f2 RNA as a messenger. We conclude that 70S ribosomes must dissociate into subunits to initiate protein synthesis with natural mRNAs.     

Accessibility of guanine at position 44 in the invariant sequence 5''CCG44AAC3'' of Escherichia coli 5S RNA to reaction with kethoxal.

The reaction of Escherichia coli ribosomes with beta-ethoxy-alpha-ketobutyraldehyde (kethoxal) in a buffer containing 50--100 mM Tris.HCl at pH 7.4, 50 mM NH4Cl, and 5 mM Mg(OAc)2 readily released the 5S RNA from the ribosomes. When liberated, the 5S RNA is in a conformation such that position 44 is selectively reactive, in addition to the normally reactive quanines at positions 41 and 13. Positions 41 and 13 have been previously shown to react in the 5S RNA in situ. The resulting new RNase T1 resistant oligonucleotides 5'CCG 44K AAUCAG51(3') and 5'ACCCCAUG 41KCCG 44KAACUCAG51(3') have been isolated and identified. These oligonucleotides have not been found in RNase T1 digests of 5S RNA that is not released from the ribosome. The guanine at position 44 is part of the invariant sequence 5'CCG44AAC3' which includes that portion of the molecule thought to interact with the invariant 5'GT psi C3' of tRNAs in the ribosomal A site. This invariant sequence of the 5S RNA may also form part of the binding site for protein L5.