N. A. Demeshkina

The mRNA Codon Environment at the P and E Sites of Human Ribosomes Deduced from Photocrosslinking with pUUUGUU Derivatives

Three mRNA analogs—derivatives of hexaribonucleotide pUUUGUU comprising phenylalanine and valine codons with a perfluoroarylazido group attached to the C5 atom of the uridine residue at the first, second, or third position—were used for photocrosslinking with 80S ribosomes from human placenta. The mRNA analogs were positioned on the ribosome with tRNA recognizing these codons: UUU was at the P site if tRNAPhe was used, while tRNAVal was used to put there the GUU codon (UUU at the E site). Thus, the crosslinking group of mRNA analog might occupy positions –3 to +3 with respect to the first nucleotide of the codon at the P site. Irradiation of the complexes with mild UV light (λ > 280 nm) resulted in the crosslinking of pUUUGUU derivatives with 18S RNA and proteins in the ribosome small subunit. The crosslinking with rRNA was observed only in the presence of tRNA. The photoactivatable group in positions –1 to +3 binds to G1207, while that in positions –2 or –3 binds to G961 of 18S RNA. In all cases, we observed crosslinking with S2 and S3 proteins irrespective of the presence of tRNA in the complex. Crosslinking with S23 and S26 proteins was observed mainly in the presence of tRNA when modified nucleotide occupied the +1 position (for both proteins) or the –3 position (for S26 protein). The crosslinking with S5/S7 proteins was substantial when modified nucleotide was in the –3 position, this crosslinking was not observed in the absence of tRNA.

The ribosomal A site-bound sense and stop codons are similarly positioned towards the A1823–A1824 dinucleotide of the 18S ribosomal RNA

Positioning of the mRNA codon towards the 18S ribosomal RNA in the A site of human 80S ribosomes has been studied applying short mRNA analogs containing either the stop codon UAA or the sense codon UCA with a perfluoroaryl azide group at the uridine residue. Bound to the ribosomal A site, a modified codon crosslinks exclusively to the 40S subunits under mild UV irradiation. This result is inconsistent with the hypothesis [Ivanov et al. (2001) RNA 7, 1683–1692] which requires direct contact between the large rRNA and the stop codon of the mRNA as recognition step at translation termination. Both sense and stop codons crosslink to the same A1823/A1824 invariant dinucleotide in helix 44 of 18S rRNA. The data point to the resemblance between the ternary complexes formed at elongation (sense codon·aminoacyl‐tRNA·AA dinucleotide of 18S rRNA) and termination (stop codon·eRF1·AA dinucleotide of 18S rRNA) steps of protein synthesis and support the view that eRF1 may be considered as a functional mimic of aminoacyl‐tRNA.

Arrangement of the Sense and Stop Codons of the Template in the A Site of the Human Ribosome as Inferred from Crosslinking with Oligonucleotide Derivatives

Oligoribonucleotide derivatives containing Phe codon UUC along with a 3′-flanking sense or stop codon with a perfluoroarylazido group at G or U were used to study the positioning of each nucleotide of the latter codon relative to the 18S rRNA in the A site of the 80S ribosome. To place the modified sense or stop codon in the A site, tRNAPhe cognate to UCC was bound in the P site. Regardless of the position in the sense or stop codon, the modified nucleotide crosslinked with invariant dinucleotide A1823/A1824 and nucleotide A1825 in helix 44 close to the 3′ end of the 18S rRNA. Located in the second or third position of either codon, the modified G bound with invariant nucleotide G626, which is in the evolutionarily conserved 530 stem–loop fragment. The results were collated with the X-ray structure of the bacterial ribosome, and the template codon was assumed to be similarly arranged relative to the small-subunit rRNA in the ribosomal A site of various organisms.

Environment of the 5′-terminal nucleotide of the mRNA codon at the P and E sites of human ribosome: Crosslinking with pUUUGUU derivatives bearing a photoactivatable group at an uracil residue or 5′-phosphate

Photoaffinity crosslinking was carried out between 80S ribosomes from human placenta and mRNA analogs, namely, derivatives of hexaribonucleotide pUUUGUU (comprising Phe and Val codons) with a perfluoroarylazido group at the C5 atom of the uracil residue at the first position, or at the 5′-terminal phosphate. Three types of ribosome complex with 5′-32P-labeled derivatives of pUUUGUU were studied: (1) with Phe-tRNAPhe and codon UUU at the P site; (2) with tRNAPhe and codon UUU at the P site and PheVal-tRNAVal and codon GUU at the A site; (3) with Val-tRNAVal and codon GUU at the P site (codon UUU at the E site). Upon mild UV irradiation (>280 nm) of the complexes, the pUUUGUU derivatives were crosslinked to 18S rRNA and proteins in the ribosomal small subunit. In the absence of tRNA, no modification of ribosomes occurred. Nucleotides of 18S rRNA crosslinked to the mRNA analogs were identified using the reverse transcriptase analysis. It turned out that the photoactivatable group at the first nucleotide of codon pUUU at the P site is only crosslinked with G-1207 of 18S rRNA, whether this group is at the 5′-phosphate or the C5 atom of the uracil residue. If codon UUU is located at the E site, the pUUUGUU derivative with the photoactivatable group at the uracil residue modifies G-961 of 18S rRNA, which is for the first time found at the mRNA-binding center of 80S ribosomes.

The First Nucleotide of the Codon Located in the P Site of the 80S Ribosome Is Close to G1702 of the 18S rRNA as Revealed by Crosslinking to a pUUUGUU Derivative Containing a Perfluorophenylazido Group at the Guanine

Modification of the 18S rRNA with a pUUUGUU derivative carrying a perfluorophenylazido group at N7 of G was studied in the complex with the human 80S ribosome and Val-tRNAVal, which directs modified GUU to the P site. Reverse transcription reported modification of invariant G1702 of the 18S rRNA. On evidence of the results and the earlier data on affinity modification of the human ribosome with tetra- or heptaribonucleotide derivatives carrying an alkylating group at the 3′ end, the template was assumed to make a bend between the A- and P-site codons, which brings both codons closer to G1702 of the 18S rRNA.

Crosslinking of mRNA Analog, pUUAGUAUUUAUU Derivative with Photoactivatable Group at the Guanosine Residue, with Human 80S Ribosomes

Crosslinking of mRNA analog, dodecaribonucleotide pUUAGUAUUUAUU derivative carrying a perfluoroarylazido group at the guanine N7, was studied in model complexes with 80S ribosomes involving tRNA and in binary complex (i.e., in the absence of tRNA). It was shown that, irrespectively of complex formation conditions (13 mM Mg2+, or 4 mM Mg2+ in the presence of polyamines), the mRNA analog in binary complex with 80S ribosomes was crosslinked with sequence 1840–1849 of 18S rRNA, but in the complexes formed with participation of Phe-tRNAPhe (where the G residue carrying the arylazido group occupied position –3 to the first nucleotide of the UUU codon at the P site) the analog was crosslinked with nucleotide 1207. The presence and the nature of tRNA at the E site had no effect on the environment of position –3 of the mRNA analog. Efficient crosslinking of the mRNA analog with tRNA was observed in all studied types of complex. Modified codon GUA, when located at the E site, underwent crosslinking with both cognate valine tRNA and noncognate aspartate tRNA for which the extent of binding at the E site of 80S ribosomes was almost the same and depended little on Mg2+ concentration and the presence of polyamines.

Analog of mRNA, pUUUGUU derivative with an arylazide group at guanosine residue, crosslinks with nucleotides A1823 and A1824 of 18S rRNA in human 80S ribosomes

Affinity modification of human placental 80S ribosomes was performed using an mRNA analog, pUUUGUU derivative containing a 3′-O-methyl group and photoactivatedp-azidotetrafluorobenzoyl group at the N7 atom of guanine, in complexes with one or two codon-anticodon interactions formed with participation of Phe-tRNAPhe or Phe-tRNAPhe and Val-tRNAVal (in both cases, the modified codon GUU was at the A site). It was established by the reverse transcription technique that the mRNA analog crosslinks with nucleotides A1823 and A1824 of 18S rRNA in the complexes of both types.

Arrangement of the Template 3′ of the A-Site Codon on the Human 80S Ribosome

The arrangement of the template sequence 3′ of the A-site codon on the 80S ribosome was studied using mRNA analogs containing Phe codon UUU at the 5′ end and a photoreactive perfluoroarylazido group linked to C5 of U or N7 of G. The analogs were positioned on the ribosome with the use of tRNAPhe, which directed the UUU codon to the P site, bringing a modified nucleotide to position +9 or +12 relative to the first nucleotide of the P-site codon. Upon mild UV irradiation of ribosome complexes, the analogs of both types crosslinked to the 18S rRNA and proteins of the 40S subunit. Comparisons were made with the crosslinking patterns of complexes in which an mRNA analog contained a modified nucleotide in position +7 (the crosslinking to 18S rRNA in such complexes has been studied previously). The efficiency of crosslinking to ribosomal components depended on the nature of the modified nucleotide of an mRNA analog and its position on the ribosome. The extent of crosslinking to the 18S rRNA drastically decreased as the modified nucleotide was transferred from position +7 to position +12. The 18S rRNA nucleotides involved in crosslinking were identified. A modified nucleotide in position +9 crosslinked to the invariant dinucleotide A1824/A1825 and variable A1823 in the 3′ minidomain of the 18S rRNA and to S15. The same ribosomal components have earlier been shown to crosslink to modified nucleotides in positions +4 to +7. In addition, all mRNA analogs crosslinked to invariant C1698 in the 3′ minidomain and to conserved region 605–620, which closes helix 18 in the 5′ domain.

[Template location on the human ribosome: environment of the mRNA nucleotide adjacent to the A-site codon on the 3'-side].

The 18S rRNA nucleotides close to the template nucleotide adjacent to the 80S ribosomal A-site codon on the 3′-end (i.e., the nucleotide in position +7 relative to the first nucleotide of the P-site codon) were identified using the affinity crosslinking approach. For this purpose, the photoreactive mRNA analogues with a perfluorophenylazide group attached through various linkers to the uridine C5, 3′-terminal phosphate or guanosine N7 were used. The position of the mRNA analogues on the ribosome was preset using tRNAPhe, which recognized the phenylalanine codon directed to the P-site. An analysis of the rRNAs isolated from the irradiated complexes of 80S ribosomes showed that all the analogues are almost equally crosslinked to the 18S rRNA nucleotides we attributed to the A-site codon environment: namely, to nucleotides A1823, A1824, and A1825 of the 3′-minidomain and to the 620–630 fragment of the 18S rRNA 5′-domain. In addition, we identified a new component of the mRNA binding site of human ribosomes, nucleotide C1698, belonging to the 18S rRNA 3′-minidomain, using analogues bearing a perfluorophenylazide group on uridine and guanine residues.