Segments of 18S rRNA, located in the area of the mRNA-binding center of ribosomes from human placenta

The affinity labelling of human placenta 80S ribosomes by 4-(N-2-chloroethyl-N-methylamino)benzyl-5'-phosphoramide of hexauridylate has been studied. This mRNA analogue has normal coding properties because its binding to placenta ribosomes significantly increases in the presence of the cognate tRNA(Phe). Incubation of the mRNA analogue in the complex with ribosomes and Phe-tRNAPhe) leads to its covalent attachment exclusively to the small subunit (mainly to 18S rRNA). The reaction site has been shown by hybridisation experiments to be located within positions 975-1055 of 18S rRNA. The identified fragment is located in a highly conserved part of the small subunit rRNA domain II.     

Identification by RNA-protein cross-linking of ribosomal proteins located at the interface between the small and the large subunits of mammalian ribosomes

Protein constituents at the subunit interface of rat liver ribosomes were analysed by cross-linking with the bifunctional reagent, diepoxybutane (distance between reactive groups 4 A). Isolated 40S and 60S subunits were labelled with 125I and recombined with unlabelled complementary subunits. The two kinds of selectively labelled 80S ribosomes were treated with diepoxybutane at low concentration. Radioactive ribosomal proteins covalently attached to the rRNA of the unlabelled complementary subparticles were isolated by repeated gradient centrifugation. The RNA-bound, labelled proteins were identified by two-dimensional gel electrophoresis. The experiments showed that proteins S2, S3, S4, S6, S7, S13, and S14 in the small subunit of rat liver ribosomes are located at the ribosomal interface in close proximity to 28S rRNA. Similarly, proteins L3, L6, L7, and L8 were found at the the interface of the large ribosomal subunit in the close vicinity of 18S rRNA.     

Affinity modification of 80S ribosomes from human placenta by derivatives of tri- and hexauridylates as mRNA analogs]

Derivatives of 5'-32P]labeled (pU)3 and (pU)6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residues attached to 5'-phosphates via phosphamide bond were applied to the affinity labeling of 80S ribosomes from human placenta. The reagents had normal coding properties and were fixed in the ribosomal mRNA-binding region by codon-anticodon interaction with cognate Phe-tRNA(Rhe) at P site (in the case of (pU)3 derivative) or at both A and P sites (in the case of (pU)6 one). Both reagents were found to modify only the 40S subunit. The sites of the reagents attachment to 18S ribosomal RNA were identified by blot-hybridization of the modified 18S rRNA with restriction fragments of the corresponding rDNA. They were found to be located within positions 976-1057 for (pU)6 derivative and within 976-1164 for (pU)3 one. These sites are located presumably within highly conserved parts of the eukaryotic small subunit rRNA secondary structure.     

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

The 18S rRNA environment of the mRNA at the decoding site of human 80S ribosomes has been studied by cross-linking with derivatives of hexaribonucleotide UUUGUU (comprising Phe and Val codons) that carried a perfluorophenylazide group either at the N7 atom of the guanine or at the C5 atom of the 5'-terminal uracil residue. The location of the codons on the ribosome at A, P, or E sites has been adjusted by the cognate tRNAs. Three types of complexes have been obtained for each type derivative, namely, (1) codon UUU and Phe-tRNAPhe at the P site (codon GUU at the A site), (2) codon UUU and tRNAPhe at the P site and PheVal-tRNAVal at the A site, and (3) codon GUU and Val-tRNAVal at the P site (codon UUU at the E site). This allowed the placement of modified nucleotides of the mRNA analog at positions -3, +1, or +4 on the ribosome. Mild UV irradiation resulted in tRNA-dependent crosslinking of the mRNA analogs to the 18S rRNA. Nucleotide G961 crosslinked to mRNA position -3, nucleotide G1207 to position +1, and A1823 together with A1824 to position +4. All of these nucleotides are located in the most strongly conserved regions of the small subunit RNA structure, and correspond to nucleotides G693, G926, G1491, and A1492 of bacterial 16S rRNA. Three of them (with the exception of G1491) had been found earlier at the 70S ribosomal decoding site. The similarities and differences between the 16S and 18S rRNA decoding sites are discussed.     

Functional topography of human ribosomes as studied by affinity labeling with reactive mRNA analogs.

Derivatives of 5'-32P labeled (pU)3 an (pU)6 bearing 4-(N-2-chloroethyl-N-methylamino)benzylmethylamine residue attached to 5'-phosphate via phosphamide bond and (Up)5U[32P]pC and (Up)11U[32P]pC bearing 4-(N-2-chloroethyl-N-methylamino)benzyl residue attached to 3'-end via benzylidene bond were applied for the affinity labeling of 80S ribosomes from human placenta in the presence of a cognate tRNA. The derivatives of 32P-labeled pAUG and pAUGU3 analogous to the 5'-phosphamides of (pU)n were used for affinity labeling of 40S subunits in the presence of ternary complex eIF-2.GTP.Met-tRNA(f). The sites of the reagents' attachment to 18S ribosomal RNA were identified by blot-hybridization of the modified 18S rRNA with restriction fragments of the corresponding rDNA. They were found to be located within positions 976-1057 for (pU)6 and pAUGU3 derivatives and within 976-1164 for (pU)3 and pAUG ones. The sites of 18S rRNA modification with the derivatives of (Up)5UpC and (Up)11UpC were found within positions 1610-1869 at 3'-end of the molecule. All the sites identified here are located presumably within highly conserved parts of the eukaryotic small subunit rRNA secondary structure.     

Positioning of the mRNA stop signal with respect to polypeptide chain release factors and ribosomal proteins in 80S ribosomes

To study positioning of the mRNA stop signal with respect to polypeptide chain release factors (RFs) and ribosomal components within human 80S ribosomes, photoreactive mRNA analogs were applied. Derivatives of the UUCUAAA heptaribonucleotide containing the UUC codon for Phe and the stop signal UAAA, which bore a perfluoroaryl azido group at either the fourth nucleotide or the 3'-terminal phosphate, were synthesized. The UUC codon was directed to the ribosomal P site by the cognate tRNA(Phe), targeting the UAA stop codon to the A site. Mild UV irradiation of the ternary complexes consisting of the 80S ribosome, the mRNA analog and tRNA resulted in tRNA-dependent crosslinking of the mRNA analogs to the 40S ribosomal proteins and the 18S rRNA. mRNA analogs with the photoreactive group at the fourth uridine (the first base of the stop codon) crosslinked mainly to protein S15 (and much less to S2). For the 3'-modified mRNA analog, the major crosslinking target was protein S2, while protein S15 was much less crosslinked. Crosslinking of eukaryotic (e) RF1 was entirely dependent on the presence of a stop signal in the mRNA analog. eRF3 in the presence of eRF1 did not crosslink, but decreased the yield of eRF1 crosslinking. We conclude that (i) proteins S15 and S2 of the 40S ribosomal subunit are located near the A site-bound codon; (ii) eRF1 can induce spatial rearrangement of the 80S ribosome leading to movement of protein L4 of the 60S ribosomal subunit closer to the codon located at the A site; (iii) within the 80S ribosome, eRF3 in the presence of eRF1 does not contact the stop codon at the A site and is probably located mostly (if not entirely) on the 60S subunit.     

The mRNA codon environment at the P and E sites of human ribosomes deduced from photo crosslinking with 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.     

Crosslinking of mRNA analog, pUUAGUAUUUAUU derivative with a photoactivated 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-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.     

Study of the mRNA-binding region of ribosomes at different steps of translation. II. Affinity modification of Escherichia coli ribosomes by benzylidene derivative of AUGU6 in the 70S initiation complex

2',3'-O-(4-[N-(2-chloroethyl)-N-methylamino]) benzylidene derivative of AUGU6 was used for identification of the proteins in the region of the mRNA-binding centre of E. coli ribosomes. This derivative alkylated ribosomes (preferentially 30S ribosomal) with high efficiency within the 70S initiation complex. In both 30S and 50S ribosomal subunits proteins and rRNA were modified. Specificity of the alkylation of ribosomal proteins and rRNA with the reagent was proved by the inhibitory action of AUGU6. Using the method of two-dimensional electrophoresis in polyacrylamide gel the proteins S4, S12, S13, S14, S15, S18, S19 and S20/L26 which are labelled by the analog of mRNA were identified.     

The conserved A-site finger of the 23S rRNA: just one of the intersubunit bridges or a part of the allosteric communication pathway?

During the translocation of tRNAs and mRNA relative to the ribosome, the B1a, B1b and B1c bridges undergo the most extensive conformational changes among the bridges between the large and the small ribosomal subunits. The B1a bridge, also called the "A-site finger" (ASF), is formed by the 23S rRNA helix 38, which is located right above the ribosomal A-site. Here, we deleted part of the ASF so that the B1a intersubunit bridge could not be formed (DeltaB1a). The mutation led to a less efficient subunit association. A number of functional activities of the DeltaB1a ribosomes, such as tRNA binding to the P and A-sites, translocation and EF-G-related GTPase reaction were preserved. A moderate decrease in EF-G-related GTPase stimulation by the P-site occupation by deacylated tRNA was observed. This suggests that the B1a bridge is not involved in the most basic steps of the elongation cycle, but rather in the fine-tuning of the ribosomal activity. Chemical probing of ribosomes carrying the ASF truncation revealed structural differences in the 5S rRNA and in the 23S rRNA helices located between the peptidyltransferase center and the binding site of the elongation factors. Interestingly, reactivity changes were found in the P-loop, an important functional region of the 23S rRNA. It is likely that the A-site finger, in addition to its role in subunit association, forms part of the system of allosteric signal exchanges between the small subunit decoding center and the functional centers on the large subunit.     

Structural-functional topography of human ribosomes based on the data from crosslinking with mRNA analogs--oligoribonucleotide derivatives

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.     

Nucleotide G-1207 of 18S rRNA is an essential component of the human 80S ribosomal decoding center.

mRNA analogues-derivatives of oligoribonucleotides consisting of two different codons and bearing an aryl azide group at the 5'-phosphates-were crosslinked to human 80S ribosomes by UV-irradiation of the various model complexes obtained in the presence of the cognate tRNAs. Three sequences, namely pUUUGUU (coding for Phe and Val), pUUCUAAA (first triplet coding for Phe and second being stop-codon), and pGUGUUU (coding for Val and Phe), have been used. Sequences of 18S rRNA containing nucleotides crosslinked to the mRNA analogues were examined by hydrolysis with RNase H in the presence of various cDNA probes. Crosslinked nucleotides were identified by primer extension. In all cases, only nucleotide G-1207 (equivalent to G-926 in Escherichia coli 16S rRNA) has been detected as crosslinked. Crosslinking of the mRNA analogues to the large ribosomal subunit was negligible.     

Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes.

The participation of 18S, 5.8S and 28S ribosomal RNA in subunit association was investigated by chemical modification and primer extension. Derived 40S and 60S ribosomal subunits isolated from mouse Ehrlich ascites cells were reassociated into 80S particles. These ribosomes were treated with dimethyl sulphate and 1-cyclohexyl-3-(morpholinoethyl) carbodiimide metho-p-toluene sulfonate to allow specific modification of single strand bases in the rRNAs. The modification pattern in the 80S ribosome was compared to that of the derived ribosomal subunits. Formation of complete 80S ribosomes altered the extent of modification of a limited number of bases in the rRNAs. The majority of these nucleotides were located to phylogenetically conserved regions in the rRNA but the reactivity of some bases in eukaryote specific sequences was also changed. The nucleotides affected by subunit association were clustered in the central and 3'-minor domains of 18S rRNA as well as in domains I, II, IV and V of 5.8/28S rRNA. Most of the bases became less accessible to modification in the 80S ribosome, suggesting that these bases were involved in subunit interaction. Three regions of the rRNAs, the central domain of 18S rRNA, 5.8S rRNA and domain V in 28S rRNA, contained bases that showed increased accessibility for modification after subunit association. The increased reactivity indicates that these regions undergo structural changes upon subunit association.     

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-tRNA^Phe (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.     

Bud23 methylates G1575 of 18S rRNA and is required for efficient nuclear export of pre-40S subunits

BUD23 was identified from a bioinformatics analysis of Saccharomyces cerevisiae genes involved in ribosome biogenesis. Deletion of BUD23 leads to severely impaired growth, reduced levels of the small (40S) ribosomal subunit, and a block in processing 20S rRNA to 18S rRNA, a late step in 40S maturation. Bud23 belongs to the S-adenosylmethionine-dependent Rossmann-fold methyltransferase superfamily and is related to small-molecule methyltransferases. Nevertheless, we considered that Bud23 methylates rRNA. Methylation of G1575 is the only mapped modification for which the methylase has not been assigned. Here, we show that this modification is lost in bud23 mutants. The nuclear accumulation of the small-subunit reporters Rps2-green fluorescent protein (GFP) and Rps3-GFP, as well as the rRNA processing intermediate, the 5' internal transcribed spacer 1, indicate that bud23 mutants are defective for small-subunit export. Mutations in Bud23 that inactivated its methyltransferase activity complemented a bud23Delta mutant. In addition, mutant ribosomes in which G1575 was changed to adenosine supported growth comparable to that of cells with wild-type ribosomes. Thus, Bud23 protein, but not its methyltransferase activity, is important for biogenesis and export of the 40S subunit in yeast.     

Variable and conserved elements of human ribosomes surrounding the mRNA at the decoding and upstream sites

This study is centred upon an important biological problem concerning the structural organization of mammalian ribosomes that cannot be studied by X-ray analysis because 80S ribosome crystals are still unavailable. Here, positioning of the mRNA on 80S ribosomes was studied using mRNA analogues containing the perfluorophenylazide cross-linker on either the guanosine or an uridine residue. The modi-fied nucleotides were directed to positions from −9 to +6 with respect to the first nucleotide of the P site bound codon by a tRNA cognate to the triplet targeted to the P site. Upon mild UV-irradiation, the modified nucleotides at positions +4 to +6 cross-linked to protein S15 and 18S rRNA nucleotides A1823–A1825. In addition, modified guanosines in positions +5 and +6 also cross-linked to G626, and in position +1 to G1702. Cross-linking from the upstream positions was mainly to protein S26 that has no prokaryotic homologues. These findings indicate that the tail of mammalian S15 comes closer to the decoding site than that of its prokaryotic homologue S19, and that the environments of the upstream part of mRNA on 80S and 70S ribosomes differ. On the other hand, the results confirm the widely accepted idea regarding the conserved nature of the decoding site of the small subunit rRNA.     

Structural and Functional Topography of Human Ribosomes Deduced from Crosslinking with mRNA Analogs,Oligoribonucleotide Derivatives

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 groups at different positions. A comparison of data on the template position on the human and Escherichia coliribosomes 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.     

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 tRNA^Phe, 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.