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.

Affinity labeling of 80S ribosomes from human placenta has been studied using various mRNA analogs, namely, 2',3'-O-[4-(N-2-chloroethyl)-N-methylamino]benzylidene derivatives of oligoribonucleotides (Up)(n-1)U[32P]pC (n = 3, 6 or 12) and AUGU3[32P]pC as well as ([4-(N-2-chloroethyl)-N-methylamino]benzylmethyl-[5'-32P]-phospham ide derivatives of pAUGUn (n = 3 or 6). Labeling of 80S ribosomes with the derivatives of oligouridylates was carried out in complexes obtained nonenzymatically in the presence of saturating amounts of Phe-tRNA(Phe). Complexes with derivatives bearing AUG codon were obtained using a fractionated lysate from rabbit reticulocytes which contained protein translation factors and was deprived from endogeneous ribosomes and mRNAs. In all cases, 40S subunits were labeled preferentially. Within the subunits, both 18S rRNA and proteins were found to be modified. Sites of cross-linking in 18S rRNA have been identified earlier. Here, it is shown that the main targets of cross-linking among the ribosomal proteins were S3 and S3a (with minor modification of S26) for the 3'-derivatives of (Up)5UpC and (Up)11UpC. For the same derivative of (Up)2UpC, the reverse modification pattern was observed. 5'-derivatives of pAUGUn were cross-linked to proteins S3 and S3a in comparable extent; 3'-derivative of AUGU3pC modified protein S3a preferentially.

Studying functional significance of the sequence 980-1061 in the central domain of human 18S rRNA using complementary DNA probes.

Region 980-1061 in human 18S rRNA has been chosen on the basis of our previous results, indicating that cross-linking sites of the alkylating mRNA analogs are located within this region. In the present study, we have used 10 DNA 15-mers complementary to various overlapping sequences within the 18S rRNA positions 980-1061. Their abilities to bind selectively to the target rRNA sequences were proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Four sequences (980-994, 987-1001, 1025-1039 and 1032-1046) were found to be well accessible for binding of the respective cDNA probes within 40S subunits. None of the oligomers inhibited tRNA(Phe)-dependent binding of oligo(U) messenger to 40S subunits and binding of Met-tRNA(imet) to 40S subunits in the presence of eIF-2 and nonhydrolysable GTP analog. Nevertheless, two probes (complementary to the 18S rRNA sequences 987-1001 and 1025-1039) being covalently attached to 40S subunits, inhibited translation of poly(U) by human 80S ribosomes in a cell-free system. The same oligomers revealed the most pronounced inhibitory action on the binding of messenger trinucleotide in the complex pAUG.40S.Met-tRNA(imet).eIF-2.GTP. Results of these functional assays demonstrate the importance of the 18S rRNA sequences 987-1001 and 1025-1039 for translation process on human ribosomes, most probably at the initiation step.

[Functional value of 980-1061 sequences of human 18S ribosomal RNA using complementary DNA probes]. / Izuchenie funktsional'noi znachimosti posledovatel'nosti 980-1061 16S ribosomnoi RNA cheloveka s pomoshch'iu komplemtarnykh DNK-zondov.

Region 980-1061 in human 18S rRNA was chosen on the basis of our previous results indicating, that the cross-linking sites of alkylating mRNA analogs are located within this region. In the present study, we have used 10 DNA 15-mers complementary to various overlapping sequences within the 18S rRNA positions 980-1061. Their ability to bind selectively at the desired rRNA sequences was proved by hydrolysis of 18S rRNA within heteroduplexes with the corresponding probes by RNase H. Only four of the probes were able to bind to 40S subunits indicating, that the corresponding 18S rRNA sequences 980-994, 987-1001, 1025-1039 and 1032-1046 are exposed within the subunits. None of the probes inhibited tRNA-dependent binding of oligo(U) messengers to 40S subunits. Nevertheless, two probes (complementary to 18S rRNA sequences 987-1001 and 1025-1039) being covalently attached to 40S subunits, inhibited translation of poly(U) by human 80S ribosomes in a cell-free system. The binding of messenger trinucleotide in the complex pAUG.40S.Met-tRNA.eIF-2.GTP was strongly affected by the same oligomers. Thus 987-1001 and 1025-1039 18S rRNA sequences are supposed to be involved in interaction with mRNA in the course of translation.

[Structural elements of 80S ribosomes located near the 5'-region of the mRNA-binding center]. / Strukturnye élementy 80S ribosomy, raspolozhennye vblizi 5'-oblasti mRNK-sviazyvaiushchego tsentra.

Affinity labeling of 80S ribosomes with 4-(N-2-chloroethyl-N-methylamino)benzylmethylphosphoramides of oligoribonucleotides [32P]AUGUn--mRNA analogs--was studied in three model complexes: 80S.ClRCH2N(CH3)-pAUGU6.Met(Phe)2-trRNA(Phe), 80S.ClRCH2N(CH3)pAUGU3.MetPhe-tRNA(Phe), and 80S.ClRCH2N(CH3)-pAUG.Met- tRNA(Met). Two of these complexes imitate the posttranslocational state of 80S ribosomes. Small subunits were labeled preferentially; both 18S rRNA and ribosomal proteins were modified by the mRNA analogs. The relative modification extents of proteins and rRNA depended on the length of the reagent oligoribonucleotide moiety. Extension of the latter resulted in decrease in the relative extent of 18S rRNA modification from 95 a to 16% (for proteins, increase from 5 to 84%, respectively). Fragments of 18S rRNA containing cross-linking sites were identified using blot hybridization. In all cases, fragment 976-1164 was found to be modified. In the case of ClRCH2N(CH3)pAUGU6, labeling occurred also within fragments 593-673 and 1748-1869. Analysis of the modified proteins revealed that proteins S14/S15 were labeled with all three reagents and were the single target of modification with ClRCH2N(CH3)pAUGU6. Proteins S3/S3a, S6, and S16/S18 were modified only with ClRCH2N(CH3)pAUGU3; protein S20 only with ClRCH2N(CH3)pAUG; and proteins S5 and S17 were labeled with both reagents (n = 0, 3).

[Interaction of mRNA analogs--oligoribonucleotides AUGU3, AUGU6 and (pU)6 with 80S ribosomes from human placenta in the presence of related tRNA and a protein-synthesizing system]. / Vzaimodeistvie analogov mRNK--oligoribonukleotidov AUGU2, AUGU6, and (pU)6 s 80S ribosomami iz platsenty cheloveka v prisutstvii rodstvennykh tRNK i beloksinteziruiushchei sistemy.

Binding of oligoribonucleotides AUGUn (n-3 and 6) and (pU) to 80S ribosomes from human placenta in the presence of cognate tRNAs and a "ribosome-free" protein-synthesizing system from rabbit reticulocytes has been studied. The binding of the mRNA analogues resulted in formation of stable post-translocational complexes (which may be easily isolated by centrifugation in sucrose density gradient): 80S.AUGU3.MetPhe-tRNA(Phe); 80S.AUGU6.Met(Phe)2.tRNA(Phe); 80S.(pU)6.(Phe)2-tRNA(Phe). In these complexes the ratios of the bound ligands are close to the theoretically expected values. Comparison of the results obtained with the previously reported data on nonenzymatic binding of oligouridylates and Phe-tRNA(Phe) to 80S ribosomes lead one to the conclusion that translation factors significantly stabilize the complexes of tRNA with 80S ribosomes and oligoribonucleotide templates.

Selective inhibition of the polypeptide chain elongation in eukaryotic cells.

The effect of Cephalotaxus alkaloids--homoharringtonine and cephalotaxine--on translation in a cell-free system from rabbit reticulocytes and on phenylalanine polymerisation by human ribosomes was studied. The effect of the alkaloids on the nonenzymatic and the eEF-1-dependent Phe-tRNA(Phe) binding to poly(U)-programmed 80S ribosomes, diphenylalanine synthesis accompanying nonenzymatic Phe-tRNA(Phe) binding and acetylphenylalanyl-puromycin formation was examined. Homoharringtonine was shown to inhibit the formation of diphenylalanine and acetylphenylalanyl-puromycin catalysed by human and rat liver ribosomes, but was inactive as an inhibitor on the E. coli elongation system. Neither nonenzymatic nor enzymatic Phe-tRNA(Phe) binding was noticeably affected by the alkaloid. It has been proposed that the site of homoharringtonine binding to 80S ribosomes should overlap or coincide with the acceptor site of the ribosomal peptidyl transferase centre. The association constant of homoharringtonine for 80S human ribosomes was estimated to be (2.57 +/- 0.33).10(7) M-1 in the presence of puromycin. Cephalotaxine did not exert a significant influence on the polypeptide chain elongation.

Isolation of ribosomal subunits containing intact rRNA from human placenta: estimation of functional activity of 80S ribosomes.

We have elaborated a method for the isolation of ribosomal subunits from fresh unfrozen human placenta containing intact rRNA and a complete set of ribosomal proteins. Activity of 80S ribosomes obtained by reassociation of 40S and 60S subunits in nonenzymatic poly(U)-dependent binding of Phe-tRNA(Phe) was equal to 80% (above 1.5 mol [14C]Phe-tRNA(Phe) is coupled to 1 mol of ribosomes). The activity of 80S ribosomes in poly(U)-directed synthesis of polyphenylalanine was tested in a polysome-free protein-synthesizing system from rabbit reticulocytes. About 100 mol of phenylalanine residue was polymerized by a mole of ribosomes at a rate of 0.83 residues per minute in this system (2 h, 37 degrees C).

[Isolation of ribosomal subparticles from human placenta containing intact rRNA and determination of the functional activity of the 80S ribosome]. / Vydelenie ribosomykh subchastits iz platsenty cheloveka, soderzhashchikh intaktnuiu rRNK, i opredelenie funktsional'noi aktivnosti 80S ribosom.

The method for isolation of human placenta ribosomal subunits containing intact rRNA has been determined. The method uses fresh unfrozen placenta. Activity of 80S ribosomes obtained via reassociation of 40S and 60S subunits in non-enzymatic poly(U)-mediated Phe-tRNAPhe binding, was near 75% (maximal [14C]Phe-tRNA(Phe) binding was 1.5 mol Phe-tRNA(Phe) per mol of 80S ribosomes). Activity of 80S ribosomes with damaged rRNA isolated from frozen placenta was 2 times lower (the maximum level of poly(U)-dependent Phe-tRNA(Phe) binding was 0.7 mol per mol of ribosomes). The activity 80S ribosomes in poly(U)-mediated synthesis of polyphenylalanine was determined by using fractionated ("ribosomeless") protein synthesising system from rabbit reticulocytes. In this system up to the 50 mol of Phe residues per mol of 80S ribosomes are incorporated in acid insoluble fraction in 1 hour, at 37 degrees C. The obtained level of [14C]phenylalanine incorporation is three times as much as the amount of Phe residues observed for the ribosomal subunits, isolated from frozen placenta.

[The effect of Cephalotaxus group alkaloids on elongation of the polypeptide chain in human ribosomes]. / Vliianie alkaloidov gruppy Cephalotaxus na élongatsiiu polipeptidnoi tsepi na ribosomakh cheloveka.

Effects of Cephalotaxus alkaloids (homoharringtonine and cephalotaxine) on the translation of endogenous mRNA in a cell-free system of rabbit reticulocyte lysate and on poly(U)-directed poly(Phe) synthesis on human placenta ribosomes was studied. The effect of the alkaloids on the activity of human placenta ribosomes in a template-dependent aminoacyl-tRNA binding, N-acetyl-phenylalanyl-puromycin and diphenylalanine formation was also studied. Homoharringtonine was shown to have little effect of codon-dependent Phe-tRNA(Phe) binding but the alkaloid strongly inhibited (Phe)2 formation as well as N-Ac-Phe-puromycin synthesis from the complex N-Ac-Phe-tRNA(Phe).poly(U).80S ribosomes. It was concluded that the site of homoharringtonine binding overlaps or coincides with the acceptor site of the ribosomal peptidyltransferase center. The association constant of homoharringtonine to the ribosomes was estimated to be (4.8 +/- 1.0) x 10(7) M-1. Cephalotaxine had no effect on the elongation steps.

[Characteristics of nonenzymatic binding of oligoribonucleotides-- analogs of mRNA and Phe-tRNA Phe with 80S ribosomes from human placenta]. / Osobennosti neénzimaticheskogo sviazyvaniia oligoribonukleotidov-- analogov mRNK i Phe-tRNK Phe s 80S ribosomami iz platsenty cheloveka.

Binding of labelled oligouridylates--mRNA analogs--to human placenta 80S ribosomes in the presence of Phe-tRNAPhe has been studied. The single site for (pU)n (n = 6, 9, 13) binding on the ribosome was found; association constants for their tRNA-dependent binding were evaluated. In the presence of oligouridylates as templates [14C]Phe-tRNAPhe was found to be able to bind simultaneously at acceptor and donor ribosomal sites which resulted in diphenylalanine formation. The observed maximum Phe-tRNAPhe binding level was considerably lower than for the corresponding oligouridylate binding; the longer oligouridylate the higher Phe-tRNAPhe maximum binding level. To explain the results obtained we have proposed that (i) (Phe)2-tRNA produced from transpeptidation dissociates from the ribosomal A site to a significant extent and (ii) when oligouridylate length increases its interaction with 3'-side of mRNA binding center results in allosteric stabilization of the complex of peptidyl-tRNA with the ribosome at A site.

Structural arrangement of tRNA binding sites on Escherichia coli ribosomes, as revealed from data on affinity labelling with photoactivatable tRNA derivatives.

A systematic study of protein environment of tRNA in ribosomes in model complexes representing different translation steps was carried out using the affinity labelling of the ribosomes with tRNA derivatives bearing aryl azide groups scattered statistically over tRNA guanine residues. Analysis of the proteins crosslinked to tRNA derivatives showed that the location of the derivatives in the aminoacyl (A) site led to the labelling of the proteins S5 and S7 in all complexes studied, whereas the labelling of the proteins S2, S8, S9, S11, S14, S16, S17, S18, S19, S21 as well as L9, L11, L14, L15, L21, L23, L24, L29 depended on the state of tRNA in A site. Similarly, the location of tRNA derivatives in the peptidyl (P) site resulted in the labelling of the proteins L27, S11, S13 and S19 in all states, whereas the labelling of the proteins S5, S7, S9, S12, S14, S20, S21 as well as L2, L13, L14, L17, L24, L27, L31, L32, L33 depended on the type of complex. The derivatives of tRNA(fMet) were found to crosslink to S1, S3, S5, S7, S9, S14 and L1, L2, L7/L12, L27. Based on the data obtained, a general principle of the dynamic functioning of ribosomes has been proposed: (i) the formation of each type of ribosomal complex is accompanied by changes in mutual arrangement of proteins - 'conformational adjustment' of the ribosome - and (ii) a ribosome can dynamically change its internal structure at each step of initiation and elongation; on the 70 S ribosome there are no rigidly fixed structures forming tRNA-binding sites (primarily A and P sites).

[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]. / Issledovanie mRNK-sviazyvaiushchei ioblasti ribosomy na raznykh étapakh transliatsii. II. Affinnaia modifikatsiia ribosom Escherichia coli benzilidenovym proizvodnym AUGU6 v 70S komplekse initsiatsii.

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.

[Photoaffinity modification of Escherichia coli ribosomes near the tRNA-binding centers by tRNAPhe derivatives carrying arylazido groups on guanosine residues]. / Fotoaffinnaia modifikatsiia ribosom Escherichia coli vblizi tRNK-sviazyvaiushchikh tsentrov proizvodnymi tRNKPhe, nesushchimi arilazidogruppy na ostatkakh guanozina.

Photoreactive derivatives of tRNAPhe (E. coli) bearing arylazido groups scattered statistically over guanosine residues (azido-tRNA) were applied for affinity labelling of E. coli ribosomes in elongation factor-dependent or factor-free model systems mimicking different steps of elongation. It is shown that UV-irradiation of the corresponding complexes of ribosomes with tRNA derivatives results in labelling of both subunits, the 30S one is labelled preferentially. In all experiments only ribosomal proteins were labelled. Comparison of the sets of proteins labelled by tRNA derivatives in different states at P-site allowed us to draw important conclusions concerning the influence of peptidyl moiety and of the occupancy of the A-site with aminoacyl- or peptidyl-tRNA on the arrangement of tRNA located at the P-site. Three of the 30S proteins--S11, S13 S19--are labelled with tRNA derivatives located at P-site in all states.

[Affinity modification of Escherichia coli ribosomes near the acceptor tRNA-binding site]. / Affinnaia modifikatsiia ribosom Escherichia coli vblizi aktseptornogo tRNK-sviazyvaiushchego uchastka.

It was shown that Phe-tRNA Phe derivatives bearing arylazidogroups scattered statistically on N7 guanosine residues retain the ability to EF-Tu-dependent binding to E. coli ribosomes. UV-irradiation of the corresponding complex with the derivative of Phe-tRNA Phe located at A-site results in a specific modification of both ribosomal subunits to an approximately equal extent. It was found that proteins S9, S15, S16, S17, S18, S19 and L8/L9, L13, L15, L27 are labelled at A-site.

The effect of aminoacyl- or peptidyl-tRNA at the A-site on the arrangement of deacylated tRNA at the ribosomal P-site.

Photoreactive derivatives of E. coli tRNAPhe bearing arylazido groups on guanine residues (azido-tRNA) were used for affinity labelling of E. coli ribosomes in the region of the P-site when the A-site was either free or occupied by aminoacyl- or peptidyl-tRNA. Corresponding complexes of azido-tRNA with ribosomes and poly(U) were obtained both nonenzymatically and with the use of elongation factors. UV-irradiation of the complexes resulted in labelling of ribosomal proteins (preferentially of 30 S subunit). Proteins S9 and S21 were labelled only when the A-site was free; S14 - only when it was occupied; S11, S13, S19 - in both cases; S5, S7, S12, S20 - in some states.