[Fragment of mRNA coding part that is complementary to region 1638-1650 of wheat 18S rRNA functions as a translational enhancer].

Possible involvement of 18S rRNA fragment 1638-1650 including basements of the helices h44 and h28 and nucleotides of the ribosomal decoding site in the cap-independent translation initiation on plant ribosomes is studied. This rRNA fragment is shown to be accessible for complementary interactions within the 40S ribosomal subunit. It is found that the sequence complementary to the 18S rRNA fragment 1638-1650 is able to enhance efficiency of a reporter mRNA translation when placed just after the initiation codon. The results obtained indicate that in the course of the cap-independent translation initiation, complementary interactions can occur between mRNA coding sequence and 18S rRNA fragment in the region of the ribosomal decoding site.

[Probable involvement of 3'-terminal segment of 18S rRNA in translation initiation of uncapped mRNAs in plants].

A possibility of involvement of 3'-terminal 18S rRNA segment in the cap-independent initiation of translation on plant ribosomes was studied. It was shown that 3-terminal segment (nucleotides 1777-1811) of 18S rRNA including the last hairpin 45 is accessible for complementary interactions in 40S ribosomal subunits. Oligonucleotides complementary to this segment of rRNA when added to wheat germ cell-free protein synthesizing system were found to specifically inhibit translation of uncapped reporter mRNA coding for beta-glucuronidase, which bears in the 5'-untranslated region (UTR) a leader sequence of potato virus Y (PVY) genomic RNA possessing fragments complementary to the region 1777-1811. It was shown that a sequence corresponding to nucleotides 291-316 of PVY, which is complementary to a major portion of the 3-terminal 18S rRNA segment 1777-1808, when placed into 5'-UTR, is able to enhance translational efficiency of the reporter mRNAs. The results obtained suggest that complementary interactions between mRNA 5'-UTR and 18S rRNA 3'-terminal segment can take place in the course of cap-independent translation initiation.

[Region 1112-1123 in the central domain of 18S rRNA in 40S subunits of plant ribosomes: accessibility for complementary interactions and the functional role].

The binding of the 18S RNA of the 40S subunits of wheat germ ribosomes to an oligodeoxyribonucleotide complementary to the 1112-1123 region of the central domain of this RNA molecule has been studied. The selective binding of this oligomer to the complementary RNA fragment and the inhibition of the translation of uncapped chimeric RNA containing enhancer sequences in the 5'-untranslated region upstream of the reporter sequence coding for beta-glucuronidase has been shown in a cell-free protein-synthesizing system. The use of a derivative of the aforementioned oligomer containing an alkylating group at the 5' end allowed for the demonstration that the 1112-1123 region of 18S RNA can form a heteroduplex with the complementary sequence of the oligomer. The data obtained show that the 1112-1123 region in loop 27 of the central domain of 18S RNA of 40S ribosomal subunits is exposed on the subunit surface and probably participates in the cap-independent binding of the subunits to mRNA due to the complementary interaction with the enhancer sequences.

[Molecular environment of the IIId subdomain of the IRES element of hepatitits C virus RNA on the human 40S ribosomal subunit].

The molecular environment of the key subdomain IIId of the internal ribosome entry site (IRES) element of hepatitis C virus (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, HCV IRES derivatives bearing perfluorophenylazido groups activatable by mild UV at nucleotide G263 or A275 in the subdomain IIId stem were used. They were prepared by the complementarily addressed modification of the corresponding RNA transcript with alkylating oligodeoxynucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA. It was found that the photoreactive groups of the IRES G263 and A275 nucleotides are crosslinked to ribosomal proteins S3a, S14, and S16. For the IRES derivative with the photoreactive group in nucleotide G263, the degree of modification of proteins S14 and S16 was greater than that of S3a, whereas the derivative containing the same photoreactive group in nucleotide A275 was mainly crosslinked to proteins S3a and S14. An analysis of the data led to the conclusion that, in the binary complex of HCV IRES elements with the small subunit of the 80S ribosome, its subdomain IIId stem is located on the outer subunit surface between the head and the body next to the "beak" near the exit of mRNA from the ribosome.

[Protein S3 fragments neighboring mRNA during elongation and translation termination on the human ribosome].

Protein S3 fragments were determined that crosslink to modified mRNA analogues in positions +5 to +12 relative to the first nucleotide in the P-site binding codon in model complexes mimicking states of ribosomes at the elongation and translation termination steps. The mRNA analogues contained a Phe codon UUU/UUC at the 5'-termini that could predetermine the position of the tRNA(Phe) on the ribosome by the location of P-site binding and perfluorophenylazidobenzoyl group at a nucleotide in various positions 3' of the UUU/UUC codon. The crosslinked S3 protein was isolated from 80S ribosomal complexes irradiated with mild UV light and subjected to cyanogen bromide-induced cleavage at methionine residues with subsequent identification of the crosslinked oligopeptides. An analysis of the positions of modified oligopeptides resulting from the cleavage showed that, in dependence on the positions of modified nucleotides in the mRNA analogue, the crosslinking sites were found in the N-terminal half of the protein (fragment 2-127) and/or in the C-terminal fragment 190-236; the latter reflects a new peculiarity in the structure of the mRNA binding center in the ribosome, unknown to date. The results of crosslinking did not depend on the type of A-site codon or on the presence of translation termination factor eRF1.

[C-terminal fragment of ribosomal protein S15 is located at the decoding site of the human ribosome].

Protein S15 is a characteristic component of the mammalian 80S ribosome that neighbors mRNA codon at the decoding site and the downstream triplets. In this study we determined S15 protein fragments located close to mRNA positions +4 to +12 with respect to the first nucleotide of the P site codon on the human ribosome. For cross-linking to ribosomal protein S15, a set of mRNA was used that contained triplet UUU/UUC at the 5'-termini and a perfluorophenyl azide-modified uridine in position 3' of this triplet. The locations of mRNA analogues on the ribosome were governed by tRNAPhe cognate to the UUU/UUC triplet targeted to the P site. Cross-linked S15 protein was isolated from the irradiated with mild UV light complexes of 80S ribosomes with tRNAPhe and mRNA analogues with subsequent cleavage with CNBr that splits polypeptide chain after methionines. Analysis of modified oligopeptides resulted from the cleavage revealed that in all cases cross-linking site was located in C-terminal fragment 111-145 of protein S15 indicating that this fragment is involved in formation of decoding site of the eukaryotic ribosome.

[Protein S3 in the human 80S ribosome adjoins mRNA from 3'-side of the A-site codon].

The protein environment of mRNA 3' of the A-site codon (the decoding site) in the human 80S ribosome was studied using a set of oligoribonucleotide derivatives bearing a UUU triplet at the 5'-end and a perfluoroarylazide group at one of the nucleotide residues at the 3'-end of this triplet. Analogues of mRNA were phased into the ribosome using binding at the tRNAPhe P-site, which recognizes the UUU codon. Mild UV irradiation of ribosome complexes with tRNAPhe and mRNA analogues resulted in the predominant crosslinking of the analogues with the 40S subunit components, mainly with proteins and, to a lesser extent, with rRNA. Among the 40S subunit ribosomal proteins, the S3 protein was the main target for modification in all cases. In addition, minor crosslinking with the S2 protein was observed. The crosslinking with the S3 and S2 proteins occurred both in triple complexes and in the absence of tRNA. Within triple complexes, crosslinking with S15 protein was also found, its efficiency considerably falling when the modified nucleotide was moved from positions +5 to +12 relative to the first codon nucleotide in the P-site. In some cases, crosslinking with the S30 protein was observed, it was most efficient for the derivative containing a photoreactive group at the +7 adenosine residue. The results indicate that the S3 protein in the human ribosome plays a key role in the formation of the mRNA binding site 3' of the codon in the decoding site.

[Molecular environment of the subdomain IIIe loop of the RNA IRES element of hepatitis C virus on the human 40S ribosomal subunit].

The molecular environment of the internal ribosome entry site (IRES) element of hepatitis C viral (HCV) RNA in the binary complex with the human 40S ribosomal subunit was studied. To this end, RNA derivatives bearing mild UV-reactive perfluorophenylazide groups at nucleotide G87 in IRES domain II and at nucleotide A296 in the subdomain IIIe loop were used, which were prepared by the RNA complementarily-addressed modification with alkylating oligonucleotide derivatives. None of the RNA derivatives were shown to be crosslinked to the 18S rRNA of the 40S subunit. It was found that the photoreactive group of IRES nucleotide A296 was crosslinked to the 40S subunit S2/S3a, S5, and p40 (SOA) proteins. No protein crosslinking was observed for the RNA derivative containing the same photoreactive group in nucleotide G87. It was concluded that the subdomain IIIe loop of the HCV RNA IRES element in the complex with the 40S subunit is located on the outer subunit surface between the head and the body next to the "beak" near the entrance into the mRNA-binding channel. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.

[Positioning of mRNA 3' of the a site bound codon on the human 80S ribosome].

Short mRNA analogues carrying a UUU triplet at the 5'-termini and a perfluorophenylazide group at either the N7 atom of the guanosine or the C5 atom of the uridine 3' of the triplet were applied to study positioning of mRNA 3' of the A site codon. Complexes of 80S ribosomes with the mRNA analogues were obtained in the presence of tRNAPhe that directed UUU codon to the P site and consequently provided placement of the nucleotide with cross-linker in positions +9 or +12 with respect to the first nucleotide of the P site bound codon. Both types mRNA analogues cross-linked to the 18S rRNA and 40S proteins under mild UV-irradiation. Cross-linking patterns in the complexes where modified nucleotides of the mRNA analogues were in position +7 were analyzed for comparison (cross-linking to the 18S rRNA in such complexes has been studied previously). The efficiency of cross-linking to the ribosomal components depended on the nature of the modified nucleotide in the mRNA analogue and its position on the ribosome, extent of cross-linking to the 18S rRNA being decreased drastically when the modified nucleotide was moved from position +7 to position +12. The nucleotides of 18S rRNA cross-linked to mRNA analogues were determined. Modified nucleotides in positions +9 and +12 cross-linked to the invariant dinucleotide A1824/A1825 and to variable A1823 in the 3'-minidomain of 18S rRNA as well as to protein S15. The same ribosomal components have been found earlier to cross-link to modified mRNA nucleotides in positions from +4 to +7. Besides, all mRNA analogues cross-linked to the invariant nucleotide c1698 in the 3'-minidomain and to and the conserved region 605-620 closing helix 18 in the 5'-domain.

[Part of the matrix on the 5' side from codon in the E-segment is close to protein S26 on the human 80S ribosome]. / Chast' matritsy s 5'-storony ot kodona v E-uchastke sblizhena s belkom S26 na 80S ribosome cheloveka.

Positioning of mRNA on the 80S ribosome upstream the E site bound codon was studied using derivatives of nona- and dodecaribonucleotides containing the triplet UUU coding for Phe at the 3'-terminus, and a perfluorophenylazide cross-linker on either the first or the third nucleotide. Two sets of the mRNA analogues were used, with the photoactivatable groups on either the C5 atom of the uridine or the N7 atom of the guanosine. The modified nucleotides were directed to positions from -4 to -9 with respect to the first nucleotide of the P site bound codon by tRNA(Phe) cognate to the triplet UUU targeted to the P site. Mild UV-irradiation of ribosomecomplexes with tRNA(Phe) and mRNA analogues resulted in the cross-linking to the 40S subunits preferentially, mainly to the proteins. The principal target for the cross-linking was protein S26 in all cases. Location of the photoactivatable group on the nucleotide at position -4 lead also to the minor cross-linking to protein S3, and at position -6 to protein S14. In the absence of tRNA, all mRNA analogues cross-linked to protein S3.

[Protein environment of the sense codon of the template in the A site of the human ribosome as inferred from crosslinking to oligoribonucleotide derivatives]. / Belkovoe okruzhenie smyslovogo kodona matritsy v A-uchastke ribosom cheloveka po dannym fotosshivok s proizvodnymi oligoribonukloeotidov.

The protein environment of each nucleotide of the template codon located in the A site of the human ribosome was studied with UUCUCAA and UUUGUU derivatives containing a Phe codon (UUC and UUU, respectively) and a perfluoroarylazido group at U4, U5, or U6. The analogs were positioned in the ribosome with the use of tRNA(Phe), which is cognate to the UUC or UUU codon and directs it to the P site, bringing a modified codon in the A site with a modified nucleotide occupying position +4, +5, or +6 relative to the first nucleotide of the P-site codon. On irradiation of ribosome complexes with tRNA(Phe) and mRNA analogs with mild UV light, the analogs crosslinked predominantly to the 40S subunit, modifying the proteins to a greater extent than the rRNA. The 18S rRNA nucleotides crosslinking to the analogs were identified previously. Of the small-subunit proteins, S3 and S15 were the major targets of modification in all cases. The former was modified both in ternary complexes and in the absence of tRNA, and the latter, only in ternary complexes. The extent of crosslinking of mRNA analogs to S15 decreased when the modified nucleotide was shifted from position +4 to position +6. The results were collated with the data on ribosomal proteins located at the decoding site of the 70S ribosome, and conclusion was made that the protein environment of the A-site codon strikingly differs between bacterial and eukaryotic 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]. / Pervyi nukleotid kodona v P-uchastke 80S ribosom sblizhen s nukleotidom G1702 18S rRNA--po dannym sshivok s nukleotidom 18S rRNK--po dannym sshivok s proizvodnym pUUUGUU, nesushchim perftorfenilazidogruppu na ostatke guanina.

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-tRNA(Val), 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.

[Highly conserved region 1816-1831 of the 18S ribosomal RNA is close to the first nucleotide of the A-site codon in elongation and termination of translation]. / Vysokokonservativnyi uchastok 1816-1831 18S ribosomnoi RNK sblizhen s pervym nukleotidom kodona v A-uchastke ribosomy pri élongatsii i terminatsii transliatsii.

Two mRNA analogs, pUUCUAAA (with stop codon UAA) and pUUCUCAA (with Ser codon UCA) containing a perfluoroarylazido group at U4, were used to study the position relative to the 18S rRNA for the first nucleotide of the codon located in the A site of the human 80S ribosome. To place UAA or UCA in the A site, UCC-recognizing tRNAPhe was bound in the P site. With each analog, crosslinking was detected for highly conserved fragment 1816-1831, which contains invariant dinucleotide A1823/A1824 and is in helix 44 at the 3' end of the 18S rRNA. Since 18S rRNA modification did not depend on whether the U4 photoreactive group was in the sense or stop codon, it was assumed that polypeptide chain release factor 1 directly recognizes the trinucleotide of a stop codon located in the A site.

[The mRNA codon environment at the P and E sites of human ribosomes deduced from photo crosslinking with pUUUGUU]. / Okruzhenie kodonov mRNK v P- i E-uchastkakh ribosom cheloveka po dannym fotosshivok s proizvodnymi pUUUGUU.

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 tRNA(Phe) was used, while tRNA(Val) 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 soft UV light (lambda > 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.

[Approach to identifying the functionally important segments of RNA, based on complementation-addressed modification]. / Podkhod k vyiavleniiu funktsional'no vazhnykh uchastkov RNK, osnovannyi na komplementarno-adresovannoi modifikatsaii.

An approach based on complementation-addressed modification of nucleic acids by oligodeoxyribonucleotide derivatives was proposed for changing the spatial structure of particular RNA sites in order to study their role in the biological activity of the total RNA molecule. Hepatitis C virus (HCV) IRES was used as a model. Oligodeoxyribonucleotide derivatives contained a 4-[N-(2-chloroethyl)-N-methylamino]benzylamino group at the 5'-P and were complementary to various RNA sites located in regions of hairpins II, IIId, or IIIe. Covalent adducts resulting from RNA alkylation with the derivatives were isolated by denaturing PAGE and tested for binding with the 40S subunit of human ribosomes. Structural alteration of hairpin II had no effect, whereas alteration of hairpin IIIe substantially reduced the binding. The RNA with modified hairpin IIId showed virtually no binding with the 40S subunit. Hairpin IIId was assumed to play a critical role in the binding of HCV IRES with the 40S subunit.

[Arrangement of the sense and terminating codons of the template in the A-segment of human ribosomes from photocrosslinking data withe oligonucleotide derivatives]. / Raspolozhenie smyslovogo i terminiruiushchego kodonov matritsy v A-uchastke ribosom cheloveka po dannym fotosshivok i proizvodnymi oligoribonukleotidov.

Oligoribonucleotide derivatives containing Phe codon UUC along with a 3'-flanking sense codon or stop codon carrying a perfluoroarylazido group at G or U were used to study the position 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, UCC-recognizing tRNA(Phe) 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 or 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 segment. 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 various organisms.

[Crosslinking of mRNA analog, pUUAGUAUUUAUU derivative with a photoactivated group at the guanosine residue, with human 80S ribosomes]. / Shivki analoga mRNK, proizvodnogo pUUAGUAUUUAUU s fotoaktiviruemoi gruppoi na ostatke guanozina, c 80S ribosomami cheloveka.

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-TPHKPhe (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.

[Structural-functional topography of human ribosomes based on the data from crosslinking with mRNA analogs--oligoribonucleotide derivatives]. / Strukturno-funktsional'naia topografiia ribosom cheloveka po dannym sshivok s analogami mRNK--proizvodnymi oligoribonukleotidov.

Reviewed are data on the position of template codons with respect to 18S rRNA and certain proteins on human ribosome obtained using a set of mRNA analogs, oligoribonucleotide derivatives carrying alkylating or photoactivatable group at different positions. A comparison of data on template position on the human and Escherichia coli ribosomes has revealed both the similarity in the structure of the mRNA-binding site of bacterial and mammalian ribosomes and the peculiarities of the functioning of mammalian (in particular, human) ribosomes. The similarity manifests itself in that the template codons at the A, P, and E sites of bacterial and human ribosomes are surrounded by similar nucleotides (occupying similar positions in the conserved regions of secondary structure) of small subunit rRNA. The template forms a loop whose foot is in proximity to the 530 stem-loop conserved region of rRNA. The specific features of mammalian ribosomes appear to be associated with their lower conformational mobility as compared with bacterial ribosomes, owing to which their interaction with the template involves a lesser number of molecular contacts.

Location of template on the human ribosome as revealed from data on cross-linking with reactive mRNA analogs.

In this review we summarize data on the location of template on the human ribosome that we obtained from cross-linking (affinity labeling) experiments using reactive mRNA analogs. Types of mRNA analogs, model complexes of these analogs with 80S ribosomes, and methods for analysis of the ribosomal components (proteins and rRNA nucleotides) cross-linked with the mRNA analogs are reviewed. From analysis of the cross-linking data, we suggest a scheme for the arrangement of mRNA on the human ribosome and compare the organization of the mRNA binding center on human and Escherichia coli ribosomes.