Protein environment of mRNA at the decoding site of 80S ribosomes from human placenta as revealed from affinity labeling with mRNA analogs – derivatives of oligoribonucleotides

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…”

“…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+…”

The polypeptide chain release factor eRF1 specifically contacts the s4UGA stop codon located in the A site of eukaryotic ribosomes

“…At variance with prokaryotes, the decoding center of eukaryotic ribosomes is still poorly characterized. So far a number of reactive mRNA analogs have been used to identify the components of human ribosomes surrounding the mRNA [23–27]. The crosslinks obtained with short oligoribonucleotides (3–13‐mers) bearing either a 5′/3′ end alkylating reagent [23,24] or a 5′ photoreactive arylazide probe [25] were shown to be fully dependent upon the presence of a cognate tRNA.…”

“…So far a number of reactive mRNA analogs have been used to identify the components of human ribosomes surrounding the mRNA [23–27]. The crosslinks obtained with short oligoribonucleotides (3–13‐mers) bearing either a 5′/3′ end alkylating reagent [23,24] or a 5′ photoreactive arylazide probe [25] were shown to be fully dependent upon the presence of a cognate tRNA. This approach made it possible to identify a number of 18S–rRNA crosslinks [23] and showed that proteins S3, S3a and S26 are located in the vicinity of the ribosomal decoding site [23,24].…”