Arrangement of mRNA at the Decoding Site of Human Ribosomes

Abstract

Affinity labeling of human placental 80S ribosomes with mRNA analogs of up to 12 uridyl residues, i.e. alkylating derivatives of oligouridylates bearing either 4-(N-2-chloroethyl-N-methylamino)benzylmethylphosphamide group at the 5'-termini or 2',3'-O-[4-(N-2-chloroethyl-N-methylamino)]benzylidene residue attached to the 3'-termini, in the presence of cognate Phe-tRNA(Phe) has been investigated. All the mRNA analogs modified only the 40S subunit. The fraction of 18S rRNA modified by the mRNA analogs with the alkylating group at the 5'-end decreased dramatically with extension of the reagent oligouridylate moiety. Nucleotides of 18S rRNA alkylated with the mRNA analogs were determined using a reverse transcription technique. For the mRNA analogs with the alkylating groups at the 3'-termini, G1702 and G1763/G1764 were identified as the cross-linking sites. The intensities of the bands corresponding to reverse transcriptase stops depended on the length of the reagent oligouridylate moieties. Cross-linking sites of the mRNA analogs with the alkylating group at the 5'-termini on 18S rRNA were A1023, C1026, C1057 and A1058 for the (pU)3 and (pU)4 derivatives and a single nucleotide C1057 for the (pU)6 one. Ribosomal protein S26 was found as the main target of modification with the same derivatives of (pU)6 and (pU)12.

Nucleotides of 18S rRNA surrounding mRNA codons at the human ribosomal A, P, and E sites: A crosslinking study with mRNA analogs carrying an aryl azide group at either the uracil or the guanine residue

“…Val with its cognate codons+ The binding data indicate that a modifying group at either the C5 atom of the 59-terminal uracil or at the N7 atom of the guanine of UUUGUU actually did not interfere significantly with involvement of the respective modified codon in the codon-anticodon interaction+ In accordance with all previous data on crosslinking of various mRNA analogs to human 80S ribosomes (Graifer et al+, 1990;Karpova et al+, 1992;Mundus et al+, 1993;Malygin et al+, 1994;Bulygin et al+, 1997aBulygin et al+, , 1997bSmolenskaya et al+, 1998), the 40S subunit was the main target for crosslinking; only the ratio of the 18S rRNA/proteins modification depended on the reagent and complex types+ It should be noted that for both mRNA analogs used in this study, yields of the crosslinking were very high (up to 65% of the bound mRNA analog)+ Similar yields of photocrosslinking have been observed for mRNA analogs with a p-azidotetrafluorobenzoic group attached to the 59-phosphates (Bulygin et al+, 1997a)+ The same photoactivable group attached to the 59-phosphates of deoxyoligoribonucleotides indicated similarly high (up to 70%) yields of the crosslinking of these derivatives to the complementary DNA sequences (Levina et al+, 1996)+ Thus, the high yield of photocrosslinking of nucleic acids derivatives that bear the p-azidotetrafluorobenzoic group is an advantage caused by the features of the crosslinking group itself+…”

“…Both types of the UUUGUU derivatives used in this study were unable either to bind or to crosslink to human 80S ribosomes without the cognate tRNAs (see Fig+ 3 and Table 1)+ This is typical for all short mRNA analogs that had been used earlier for investigation of the decoding center of human 80S ribosomes (e+g+, see Malygin et al+, 1994;Bulygin et al+, 1997aBulygin et al+, , 1997bSmolenskaya et al+, 1998)+ Therefore, crosslinking of the UUUGUU derivatives actually occurred at the ribosomal decoding center and the contribution of any unspecific (tRNA-independent) crosslinking was almost negligible+ The binding properties of the mRNA analogs depended mostly on the codon involved in the codon-anticodon interaction at the ribosomal P site; the modifying groups did not affect the binding significantly+ tRNA Phe (cognate to the UUU codon) stimulated binding of each of the UUUGUU derivatives to 80S ribosomes more effectively than tRNA Val (cognate to the GUU codon)+ The same effect had been observed ear- lier in a crosslinking study with derivatives of hexaribonucleotides that contained Phe and Val codons and a photoreactive group at the 59-phosphate (Bulygin et al+, 1997a)+ This implies that the P site of human 80S ribosome has higher affinity to tRNA Phe with the cognate codon UUU than to tRNA…”

“…To determine sequences of 18S rRNA containing crosslinked nucleotides, 18S rRNA isolated from irradiated complexes was digested with RNase H in the presence of deoxy-20-mers (cDNAs) complementary to various sequences of 18S rRNA in parallel experiments+ The cDNAs were chosen on the basis of our previous results on crosslinking of mRNA analogs to human ribosomes (Malygin et al+, 1994;Bulygin et al+, 1997a)+ Fragments of the rRNA resulting from the hydrolysis were separated by polyacrylamide gel electrophoresis (PAGE) under denaturing conditions+ Staining the gels allowed the detection of both parts of the rRNA cleaved in the presence of a definite cDNA, and radioautography of the gels indicated which part of the rRNA contained the crosslinked [ 32 P]-labeled mRNA analog+ Comparison of the results obtained with various cDNAs for each crosslinked 18S rRNA sample allowed the identification of relatively short sequence intervals that contained the crosslinking site+ Data on the analysis of crosslinked 18S rRNA isolated from irradiated complexes are given in Figures 4 and 5+ For (azidoU)UU-GUU in complex 1, the crosslinking site is located within 18S rRNA positions 1196-1216, because in the presence of cDNA 1197-1216 neither fragment of 18S rRNA that remained after the hydrolysis contained the crosslinked 32 P-labeled mRNA analog (Fig+ 4A, lane 2)+ Results with other cDNAs seen in the neighboring lanes are consistent with this identification+ The same results were obtained for complex 2 (data not shown)+ In complex 3, the crosslinking site of derivative I is located within 18S rRNA region 840-1081, as may be easily estimated from the data presented in Figure 4B+ For [59-32 P]pUUU(azidoG)UU in complex 2, the crosslinking site was initially found in the 39-terminal region (about 230 nt long) of 18S rRNA, as RNase H digestion to position 1812+ On the other hand, the 39-terminal sequence with the crosslinked labeled derivative II that would be formed if the crosslinking site was 39 to position 1831 was not detected+ Therefore, the crosslinking site was located within positions 1812-1831+ The same experiments for complex 1 showed that the crosslinked site was also located within the sequence 1812-1831 of 18S rRNA (data not shown)+…”

“…Results obtained in this study demonstrate the 18S rRNA environment of the first nucleotide of mRNA codon at the A, P, and E sites; the data are summarized in Table 2+ Crosslinks of mRNA analogs that bear a photoreactive group at the 59-phosphate in complexes analogous to complex 1 (the reactive group at position ϩ1) to G1207 of human 18S rRNA were detected earlier (Bulygin et al+, 1997a)+ Thus, crosslinking to G1207 from mRNA position ϩ1 does not depend on the point of attachment of the reactive group in the modified nucleotide, and G1207 is a key element of the ribosomal environment of the first nucleotide of the P site-bound codon+ It should be noted here that a derivative of pUUUUUU that carried an alkylating group at the 59-terminus was found to crosslink to 18S rRNA nucleotide C1057 in complexes with human 80S ribosomes and tRNA Phe molecules at both the A and P sites (Malygin et al+, 1994)+ In this case, the alkylating group was at mRNA position ϩ1, but crosslinking occurred at C1057 rather than at G1207, most probably because of the different chemical and/or stereochemical selectivity of the reactive intermediates formed by the aryl azide and the alkylating group (discussed in Bulygin et al+, 1997a)+ Crosslinking to 18S rRNA nucleotides A1823 and A1824 was not detected earlier+ The mRNA analog UUUC with an alkylating group at the 39-terminus was found earlier to crosslink to 18S rRNA nucleotides G1702 and G1763/G1764 in a complex with human 80S ribosomes and Phe-tRNA Phe at the P site (Malygin et al+, 1994)+ In the latter case, the position of the reactive group on the 80S ribosome was ϩ4 (if codon UUU was base paired with Phe-tRNA Phe ) or ϩ3 (codon UUC base paired with Phe-tRNA Phe )+ Data on the crosslinking of a reactive group at mRNA analog position ϩ4 to 18S rRNA nucleotides A1823 and A1824 (results of this study) and G1702 (Malygin et al+, 1994) are in a good agreement, because both 18S rRNA sites are located close to each other in a region of 18S rRNA secondary structure (see Fig+ 7), which corresponds to the "16S rRNA decoding site" (Van Loock et al+, 1999;Yoshizawa et al+, 1999)+ Nucleotide G1207, which crosslinks to a modified mRNA nucleotide at position ϩ1, is also located in the vicinity of the region mentioned (Fig+ 7)+ Crosslinking of mRNA analogs to 18S rRNA nucleotide G961 from position Ϫ3 has also been detected for the first time+ In our previous work, synthetic mRNAs that contained 4-thiouridines at positions Ϫ1 and Ϫ3 crosslinked to human 18S rRNA nucleotides U1111/ A1112 (Graifer et al+, 1994b)+ However, the crosslinking was tRNA independent, and this may be the reason for the discrepancy with the results of the present study+ Functional conservation at the small subunit rRNA decoding site…”

“…The interaction of mRNA codons with anticodons of the correct (cognate) tRNAs is one of the key steps of protein synthesis on the ribosome (translation)+ Data on the arrangement of mRNA at the site of codonanticodon interactions (the decoding site) are of great importance for understanding the molecular mechanisms of translation+ Significant progress in studying the mRNA binding center has been achieved with the use of site-directed crosslinking+ This approach is based on the application of mRNA analogs that bear chemically reactive groups or photoreactive groups at defined positions in the RNA sequence+ Numerous data on nucleotides of ribosomal RNA and ribosomal proteins crosslinked with mRNA analogs have been accumulated within the past 10 years, both for prokaryotic (mostly Escherichia coli; for a review, see Baranov et al+, 1999) and eukaryotic (in particular, human; e+g+, see Malygin et al+, 1994;Bulygin et al+, 1997aBulygin et al+, , 1997bMatasova et al+, 1998) ribosomes+ Comparison of the crosslinking data obtained with the same synthetic mRNAs (about 50 nt long) substituted randomly with 4-thiouridines revealed significant differences in interaction of mRNA with 70S and 80S ribosome (Graifer et al+, 1994a)+ For human 80S ribosomes, the only major target for crosslinking, U630, was found (Graifer et al+, 1994a) in contrast to the experiments with E. coli 70S ribosomes, where up to 12 mRNA-16S rRNA cross-links have been determined (Bhangu & Wollenzien, 1992)+ The general conclusions on the arrangement of the mRNA on the ribosome may be drawn from the sum of data obtained with the use of mRNA analogs of various natures, because results of the crosslinking may depend both on the position of the modified mRNA nucleotide on the ribosome and on the nature of the nucleotide and type of the crosslinking group (Vladimirov et al+, 1990;Malygin et al+, 1994;Bulygin et al+, 1997b;Sergiev et al+, 1997)+ Data obtained with the use of mRNA analogs that carry photoreactive groups at the positions not involved in the Watson-Crick base pairing (e+g+, C5 atom of uracil or N7 atom of guanine) are especially important+ Such analogs allow the study of the ribosomal environment of the mRNA nucleotides directly involved in the codon-anticodon interactions on the ribosome+…”