Functional involvement of a conserved motif in the middle region of the human ribosomal protein eL42 in translation.

Ribosomal protein eL42 (formerly known as L36A), a small protein of the large (60S) subunit of the eukaryotic ribosome, is a component of its exit (E) site. The residue K53 of this protein resides within the motif QSGYGGQTK mainly conserved in eukaryotes, and it is located in the immediate vicinity of the CCA-terminus of the ribosome-bound tRNA in the hybrid P/E state. To examine the role of this eL42 motif in translation, we obtained HEK293T cells producing the wild-type FLAG-tagged protein or its mutant forms with either single substitutions of conserved amino acid residues in the above motif, or simultaneous replacements in positions 45 and 51 or 45 and 53. Examination of the level of exogenous eL42 in fractions of polysome profiles from the target protein-producing cells by the Western blotting revealed that neither single substitution affects the assembly of 60S ribosomal subunits and 80S ribosomes or critically decreases the level of polysomes, but the latter was observed with the double replacements. Analysis of tRNAs bound to 80S ribosomes containing eL42 with double substitutions and examination their peptidyl transferase activity enabled estimation the stage of the elongation cycle, in which amino acid residues of the conserved eL42 motif are involved. We clearly show that cooperative interactions implicating the eL42 residues Q45, Q51, and K53 play a critical role in the ability of the human ribosome to perform properly elongation cycle at the step of deacylated tRNA dissociation from the E site in the human cell.

The functional role of the eukaryote-specific motif YxxPKxYxK of the human ribosomal protein eS26 in translation.

The protein eS26 is a structural component of the eukaryotic small ribosomal subunit involved in the formation of the mRNA binding channel in the region of the exit site. By applying site-directed cross-linking to mammalian 80S ribosomes, it has been shown that the same mRNA nucleotide residues are implicated in the interaction with both eS26 and translation initiation factor 3 (eIF3) and that contacts of the protein with mRNAs are mediated by its eukaryote-specific motif YxxPKxYxK. To examine the role of eS26 in translation, we transfected HEK293T cells with plasmid constructs encoding the wild-type FLAG-labeled protein (wt-eS26FLAG) or its forms with either a single substitution of any conserved amino acid residue in the above motif, or a simultaneous replacement of all the five ones (5A). The western blot analysis of fractions of polysome profiles from the transfected cells revealed no effects of the single mutations in eS26, but showed that the replacement of the five conserved residues led to the increased share of the light polysome fraction compared to that detected with control, wt-eS26FLAG-producing cells. In addition, the above fraction exhibited the enhanced content of the eIF3e subunit that is known to promote selective translation. These findings, together with real-time PCR data on the relative contents of specific mRNAs in light and heavy polysomes from cells producing the mutant 5A compared to those from control cells, suggest a possible involvement of the YxxPKxYxK motif of eS26 in the fine regulation of translation to maintain the required balance of synthesized proteins.

Two alternative conformations of mRNA in the human ribosome during elongation and termination of translation as revealed by EPR spectroscopy.

The conformation of mRNA in the region of the human 80S ribosome decoding site was monitored using 11-mer mRNA analogues that bore nitroxide spin labels attached to the terminal nucleotide bases. Intramolecular spin-spin distances were measured by DEER/PELDOR spectroscopy in model complexes mimicking different states of the 80S ribosome during elongation and termination of translation. The measurements revealed that in all studied complexes, mRNA exists in two alternative conformations, whose ratios are different in post-translocation, pre-translocation and termination complexes. We found that the presence of a tRNA molecule at the ribosomal A site decreases the relative share of the more extended mRNA conformation, whereas the binding of eRF1 (alone or in a complex with eRF3) results in the opposite effect. In the termination complexes, the ratios of mRNA conformations are practically the same, indicating that a part of mRNA bound in the ribosome channel does not undergo significant structural alterations in the course of completion of the translation. Our results contribute to the understanding of mRNA molecular dynamics in the mammalian ribosome channel during translation.

AP sites in various mRNA positions cross-link to the protein uS3 in the translating mammalian ribosome.

Abasic (AP) sites in mRNAs are lesions whose accumulation in cells is linked to various neurodegenerative diseases arising from the appearance of truncated peptides due to the premature cessation of translation of these mRNAs. It is believed that the translation of AP site-containing mRNAs is stopped when the damaged codon arrives to the A site, where it is not decoded. We propose an alternative translation arrest mechanism mediated by the 40S ribosomal subunit protein uS3. Recently, it has been shown that in human 80S ribosomal complexes assembled without translation factors, uS3 cross-links to the AP site at the 3'-terminus of the mRNA, whose undamaged part is bound at the 40S subunit channel, via its peptide 55-64 exposed near the mRNA entry pore. In this study, we examined whether such cross-linking occurs during the translation of mRNA with the AP site. To this end, we used a set of synthetic mRNAs bearing the AP site inserted in the desired location in their sequences. An analysis of 80S ribosomal complexes formed with these mRNAs in a mammalian cell-free protein-synthesizing system demonstrates that AP sites do indeed cross-link to uS3 in the course of the translation. We also show that the cross-linking occurs as soon as the AP site arrives to a common favorable position relative to uS3, which is independent on its location in the mRNA. Our findings suggest that the mechanism of stopping translation of damaged mRNAs involving uS3, along with the one mentioned above, could underlie ribosome-associated mRNA quality control.

Exploring the interactions of short RNAs with the human 40S ribosomal subunit near the mRNA entry site by EPR spectroscopy.

The features of previously unexplored labile complexes of human 40S ribosomal subunits with RNAs, whose formation is manifested in the cross-linking of aldehyde derivatives of RNAs to the ribosomal protein uS3 through its peptide 55-64 located outside the mRNA channel, were studied by EPR spectroscopy methods. Analysis of subatomic 40S subunit models showed that a likely site for labile RNA binding is a cluster of positively charged amino acid residues between the mRNA entry site and uS3 peptide 55-64. This is consistent with our finding that the 3'-terminal mRNA fragment hanging outside the 40S subunit prevents the cross-linking of an RNA derivative to this peptide. To detect labile complexes of 40S subunits with RNA by DEER/PELDOR spectroscopy, an undecaribonucleotide derivative with nitroxide spin labels at terminal nucleotides was utilized. We demonstrated that the 40S subunit channel occupancy with mRNA does not affect the RNA derivative binding and that uS3 peptide 55-64 is not involved in binding interactions. Replacing the RNA derivative with a DNA one revealed the importance of ribose 2'-OH groups for the complex formation. Using the single-label RNA derivatives, the distance between the mRNA entry site and the loosely bound RNA site on the 40S subunit was estimated.

The human ribosome can interact with the abasic site in mRNA via a specific peptide of the uS3 protein located near the mRNA entry channel.

The small subunit ribosomal protein uS3 is a critically important player in the ribosome-mRNA interactions during translation and has numerous functions not directly related to protein synthesis in eukaryotes. A peculiar feature of the human uS3 protein is the ability of its fragment 55-64 exposed on the 40S subunit surface near the mRNA entry channel to form cross-links with 3'-terminal dialdehyde derivatives of various unstructured RNAs and with abasic sites in single-stranded DNAs. Here we showed that the ability of the above uS3 fragment to cross-link to abasic sites in DNAs is inherent only in mature cytoplasmic 40S subunits, but not nuclear pre-40S particles, which implies that it may be relevant to the ribosome-mRNA interplay. To clarify this issue, we investigated interactions of human ribosomes with synthetic mRNA analogues bearing an abasic site protected by a photocleavable group at the 3'-termini. We found that these mRNA analogues can form specific complexes with 80S ribosomes and 40S subunits, where the undamaged upstream part of the analogue is fixed in the mRNA binding channel by interaction with the P-site tRNA, and the downstream part located outside the ribosome is cross-linked to the uS3 fragment 55-64. The yield of cross-links of the mRNA analogues was rather high when their undamaged parts were bound to the mRNA channel prior to deprotection of the abasic site enabling its covalent attachment to the 40S subunit via the uS3 protein, but not vice versa. Based on our findings, one can assume that abasic sites, which can occur in mRNAs due to oxidative stress and ageing, are able to interact directly with the uS3 fragment exposed on the 40S subunit surface near the mRNA entry channel during translation. Consequently, the 40S subunit can be considered as a potential mRNA quality controller.

Arrangements of nucleotides flanking the start codon in the IRES of the hepatitis C virus in the IRES binary complex with the human 40S ribosomal subunit.

Genomic RNA of hepatitis C virus (HCV) has an internal ribosome entry site (IRES), a specific highly structured fragment responsible for its non-canonical translation initiation. The HCV IRES contains a major part of the 5'-untranslated region of the viral RNA and a small portion of the open reading frame (ORF). At the first step of initiation, IRES directly binds to 40S ribosomal subunits so that the AUG start codon appears at the P site region without scanning and without involving initiation factors. However, it is still not entirely clear whether the IRES ORF is correctly loaded into the 40S ribosomal mRNA binding channel in the resulting binary complex. To address this issue, we applied site-directed cross-linking using HCV IRES derivatives bearing a perfluorophenyl azide cross-linker at nucleotides in definite positions relative to the adenine of the AUG start codon. We found that the modifier at the IRES position -3 cross-links to ribosomal proteins uS11 and eS26. These proteins have been identified together with uS7 as those interacting with the mRNA nucleotide in position -3 relative to the first nucleotide of the codon directed to the P site by a cognate tRNA. Thus, our results indicate a certain difference in the locations of the above parts of HCV IRES and canonical mRNAs on 40S subunits. The modifier at the IRES positions +4/5 was attached to uS19, which is specific for ribosomal complexes with the P site tRNA and similar derivatives of model canonical mRNAs when the modifier is in the same positions. However, the cross-linking efficiency of the IRES derivative was drastically lower than that previously observed with derivatives of model mRNAs. This implies that the IRES ORF portion is correctly loaded into the mRNA binding channel only in a tiny fraction of the binary complexes.

Structural rearrangements in mRNA upon its binding to human 80S ribosomes revealed by EPR spectroscopy.

The model mRNA (MR), 11-mer RNA containing two nitroxide spin labels at the 5'- and 3'-terminal nucleotides and prone to form a stable homodimer (MR)2, was used for Electron Paramagnetic Resonance study of structural rearrangements in mRNA occurring upon its binding to human 80S ribosomes. The formation of two different types of ribosomal complexes with MR was observed. First, there were stable complexes where MR was fixed in the ribosomal mRNA-binding channel by the codon-anticodon interaction(s) with cognate tRNA(s). Second, we for the first time detected complexes assembled without tRNA due to the binding of MR most likely to an exposed peptide of ribosomal protein uS3 away from the mRNA channel. The analysis of interspin distances allowed the conclusion that 80S ribosomes facilitate dissociation of the duplex (MR)2: the equilibrium between the duplex and the single-stranded MR shifts to MR due to its efficient binding with ribosomes. Furthermore, we observed a significant influence of tRNA bound at the ribosomal exit (E) and/or aminoacyl (A) sites on the stability of ribosomal complexes. Our findings showed that a part of mRNA bound in the ribosome channel, which is not involved in codon-anticodon interactions, has more degrees of freedom than that interacting with tRNAs.

Mitochondrial dysfunction and potential anticancer therapy.

Mitochondrial dysfunction (MDF) has been identified as an important factor in various diseases ranging from neurological disorders, to diseases of the cardiovascular system and metabolic syndromes. MDF was also found in cancer as well as in cancer predisposition syndromes with defective DNA damage response (DDR) machinery. Moreover, a recent highlight arises from the detection of MDF in eukaryotic cells upon treatment with antibiotics. In this review, we focus on recent studies of MDF in pathological conditions with a particular emphasis on the effects of various classes of antibiotics on mitochondria. Special attention is given to the role of autophagy/mitophagy in MDF and repurposing antibiotics as anticancer drugs.

Exploring contacts of eRF1 with the 3'-terminus of the P site tRNA and mRNA stop signal in the human ribosome at various translation termination steps.

Here we employed site-directed cross-linking with the application of tRNA and mRNA analogues bearing an oxidized ribose at the 3'-terminus to investigate mutual arrangement of the main components of translation termination complexes formed on the human 80S ribosome bound with P site deacylated tRNA using eRF1•eRF3•GTP or eRF1 alone. In addition, we applied a model complex obtained in the same way with eRF1•eRF3•GMPPNP. We found that eRF3 content in the complexes with GTP and GMPPNP is similar, proving that eRF3 does not leave the ribosome after GTP hydrolysis. Our cross-linking data allowed determining locations of the 3'-terminus of the P site tRNA relatively the eRF1 M domain and of the mRNA stop signal toward the N domain and the ribosomal decoding site at the nucleotide-peptide resolution level. Our results indicate that locations of these components do not change after peptide release up to post-termination pre-recycling state, and the positioning of the mRNA stop signal remains similar to that when eRF1 recognizes it. Besides, we found that in all the complexes studied eRF1 shielded the N-terminal part of ribosomal protein eS30 from the interaction with the nucleotide adjacent to stop codon observed with pre-termination ribosome free of eRFs. Altogether, our findings brought important information on contacts of the key structural elements of eRF1, tRNA and mRNA in the ribosomal complexes including those mimicking different translation termination steps, thereby providing a deeper understanding of molecular mechanisms underlying events occurring in the course of protein synthesis termination in mammals.

Recognition but no repair of abasic site in single-stranded DNA by human ribosomal uS3 protein residing within intact 40S subunit.

Isolated human ribosomal protein uS3 has extra-ribosomal functions including those related to base excision DNA repair, e.g. AP lyase activity that nicks double-stranded (ds) DNA 3΄ to the abasic (AP) site. However, the ability of uS3 residing within ribosome to recognize and cleave damaged DNA has never been addressed. Here, we compare interactions of single-stranded (ss) DNA and dsDNA bearing AP site with human ribosome-bound uS3 and with the isolated protein, whose interactions with ssDNA were not yet studied. The AP lyase activity of free uS3 was much higher with ssDNA than with dsDNA, whereas ribosome-bound uS3 was completely deprived of this activity. Nevertheless, an exposed peptide of ribosome-bound uS3 located far away from the putative catalytic center previously suggested for isolated uS3 cross-linked to full-length uncleaved ssDNA, but not to dsDNA. In contrast, free uS3 cross-linked mainly to the 5΄-part of the damaged DNA strand after its cleavage at the AP site. ChIP-seq analysis showed preferential uS3 binding to nucleolus-associated chromatin domains. We conclude that free and ribosome-bound uS3 proteins interact with AP sites differently, exhibiting their non-translational functions in DNA repair in and around the nucleolus and in regulation of DNA damage response in looped DNA structures, respectively.

Exploring accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel at various steps of translation initiation.

In this work, we studied how the accessibility of structural elements of the mammalian 40S ribosomal mRNA entry channel, ribosomal protein (rp) uS3 and helix (h) 16 of the 18S rRNA, changes upon the translation initiation. In particular, we examined the accessibility of rp uS3 for binding of unstructured RNAs and of riboses in h16 towards attack with benzoyl cyanide (BzCN) in complexes assembled in rabbit reticulocyte lysate utilizing synthetic oligoribonucleotides as well as full-length and truncated up to the initiation AUG codon hepatitis C virus IRES as model mRNAs. With both mRNA types, the rp uS3 peptide recognizing single-stranded RNAs was shown to become shielded only in those 48S preinitiation complexes (PICs) that contained eIF3j bound to 40S subunit in the area between the decoding site and the mRNA entry channel. Chemical probing with BzCN revealed that h16 in the 48S PICs containing eIF3j or scanning factor DHX29 is strongly shielded; the effect was observed with all the mRNAs used, and h16 remained protected as well in 80S post-initiation complexes lacking these factors. Altogether, the obtained results allowed us to suggest that eIF3j bound at the 48S PICs makes the rp uS3 inaccessible for binding of RNAs and this factor subunit is responsible for the decrease of h16 conformational flexibility; the latter is manifested as reduced accessibility of h16 to BzCN. Thus, our findings provide new insights into how eIF3j is implicated in ensuring the proper conformation of the mRNA entry channel, thereby facilitating mRNA loading.

Chemical footprinting reveals conformational changes of 18S and 28S rRNAs at different steps of translation termination on the human ribosome.

Translation termination in eukaryotes is mediated by release factors: eRF1, which is responsible for stop codon recognition and peptidyl-tRNA hydrolysis, and GTPase eRF3, which stimulates peptide release. Here, we have utilized ribose-specific probes to investigate accessibility of rRNA backbone in complexes formed by association of mRNA- and tRNA-bound human ribosomes with eRF1•eRF3•GMPPNP, eRF1•eRF3•GTP, or eRF1 alone as compared with complexes where the A site is vacant or occupied by tRNA. Our data show which rRNA ribose moieties are protected from attack by the probes in the complexes with release factors and reveal the rRNA regions increasing their accessibility to the probes after the factors bind. These regions in 28S rRNA are helices 43 and 44 in the GTPase associated center, the apical loop of helix 71, and helices 89, 92, and 94 as well as 18S rRNA helices 18 and 34. Additionally, the obtained data suggest that eRF3 neither interacts with the rRNA ribose-phosphate backbone nor dissociates from the complex after GTP hydrolysis. Taken together, our findings provide new information on architecture of the eRF1 binding site on mammalian ribosome at various translation termination steps and on conformational rearrangements induced by binding of the release factors.

Doubly Spin-Labeled RNA as an EPR Reporter for Studying Multicomponent Supramolecular Assemblies.

mRNAs are involved in complicated supramolecular complexes with human 40S and 80S ribosomes responsible for the protein synthesis. In this work, a derivative of nonaribonucleotide pUUCGUAAAA with nitroxide spin labels attached to the 5'-phosphate and to the C8 atom of the adenosine in sixth position (mRNA analog) was used for studying such complexes using double electron-electron resonance/pulsed electron-electron double resonance spectroscopy. The complexes were assembled with participation of tRNA(Phe), which targeted triplet UUC of the derivative to the ribosomal peptidyl site and predetermined location of the adjacent GUA triplet coding for Val at the aminoacyl (A) site. The interspin distances were measured between the two labels of mRNA analog attached to the first nucleotide of the peptidyl site bound codon and to the third nucleotide of the A site bound codon, in the absence/presence of second tRNA bound at the A site. The values of the obtained interspin distances agree with those calculated for available near-atomic structures of similar complexes of 40S and 80S ribosomes, showing that neither 60S subunit nor tRNA at the A site have a noticeable effect on arrangement of mRNA at the codon-anticodon interaction area. In addition, the shapes of distance distributions in four studied ribosomal complexes allowed conclusions on conformational flexibility of mRNA in these complexes. Overall, the results of this study are the first, to our knowledge, demonstration of double electron-electron resonance/pulsed electron-electron double resonance application for measurements of intramolecular distances in multicomponent supramolecular complexes involving intricate cellular machineries and for evaluating dynamic properties of ligands bound to these machineries.

Molecular contacts of ribose-phosphate backbone of mRNA with human ribosome.

In this work, intimate contacts of riboses of mRNA stretch from nucleotides in positions +3 to +12 with respect to the first nucleotide of the P site codon were studied using cross-linking of short mRNA analogs with oxidized 3'-terminal riboses bound to human ribosomes in the complexes stabilized by codon-anticodon interactions and in the binary complexes. It was shown that in all types of complexes cross-links of the mRNA analogs to ribosomal protein (rp) uS3 occur and the yield of these cross-links does not depend on the presence of tRNA and on sequences of the mRNA analogs. Site of the mRNA analogs cross-linking in rp uS3 was mapped to the peptide in positions 55-64 that is located away from the mRNA binding site. Additionally, in complexes with P site-bound tRNA, riboses of mRNA nucleotides in positions +4 to +7 cross-linked to the C-terminal tail of rp uS19 displaying a contact specific to the decoding site of the mammalian ribosome, and tRNA bound at the A site completely blocked this cross-linking. Remarkably, rps uS3 and uS19 were also able to cross-link to the fragment of HCV IRES containing unstructured 3'-terminal part restricted by the AUGC tetraplet with oxidized 3'-terminal ribose. However, no cross-linking to rp uS3 was observed in the 48S preinitiation complex assembled in reticulocyte lysate with this HCV IRES derivative. The results obtained show an ability of rp uS3 to interact with single-stranded RNAs. Possible roles of rp uS3 region 55-64 in the functioning of ribosomes are discussed.

Adenine and guanine recognition of stop codon is mediated by different N domain conformations of translation termination factor eRF1.

Positioning of release factor eRF1 toward adenines and the ribose-phosphate backbone of the UAAA stop signal in the ribosomal decoding site was studied using messenger RNA (mRNA) analogs containing stop signal UAA/UAAA and a photoactivatable cross-linker at definite locations. The human eRF1 peptides cross-linked to these analogs were identified. Cross-linkers on the adenines at the 2nd, 3rd or 4th position modified eRF1 near the conserved YxCxxxF loop (positions 125-131 in the N domain), but cross-linker at the 4th position mainly modified the tripeptide 26-AAR-28. This tripeptide cross-linked also with derivatized 3'-phosphate of UAA, while the same cross-linker at the 3'-phosphate of UAAA modified both the 26-28 and 67-73 fragments. A comparison of the results with those obtained earlier with mRNA analogs bearing a similar cross-linker at the guanines indicates that positioning of eRF1 toward adenines and guanines of stop signals in the 80S termination complex is different. Molecular modeling of eRF1 in the 80S termination complex showed that eRF1 fragments neighboring guanines and adenines of stop signals are compatible with different N domain conformations of eRF1. These conformations vary by positioning of stop signal purines toward the universally conserved dipeptide 31-GT-32, which neighbors guanines but is oriented more distantly from adenines.

Three distinct peptides from the N domain of translation termination factor eRF1 surround stop codon in the ribosome.

To study positioning of the polypeptide release factor eRF1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mRNA analogs, derivatives of oligoribonucleotides. The human eRF1 peptides cross-linked to these short mRNAs were identified. Cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31-33, and to lesser extent amino acids within region 121-131 (the "YxCxxxF loop") in the N domain. Hence, both regions are involved in the recognition of the purines. A cross-linker at the first uridine of the stop codon modifies Val66 near the NIKS loop (positions 61-64), and this region is important for recognition of the first uridine of stop codons. Since the N domain distinct regions of eRF1 are involved in a stop-codon decoding, the eRF1 decoding site is discontinuous and is not of "protein anticodon" type. By molecular modeling, the eRF1 molecule can be fitted to the A site proximal to the P-site-bound tRNA and to a stop codon in mRNA via a large conformational change to one of its three domains. In the simulated eRF1 conformation, the YxCxxxF motif and positions 31-33 are very close to a stop codon, which becomes also proximal to several parts of the C domain. Thus, in the A-site-bound state, the eRF1 conformation significantly differs from those in crystals and solution. The model suggested for eRF1 conformation in the ribosomal A site and cross-linking data are compatible.

mRNA 3' of the A site bound codon is located close to protein S3 on the human 80S ribosome.

Ribosomal proteins neighboring the mRNA downstream of the codon bound at the decoding site of human 80S ribosomes were identified using three sets of mRNA analogues that contained a UUU triplet at the 5' terminus and a perfluorophenylazide cross-linker at guanosine, adenosine or uridine residues placed at various locations 3' of this triplet. The positions of modified mRNA nucleotides on the ribosome were governed by tRNA(Phe) cognate to the UUU triplet targeted to the P site. Upon mild UV-irradiation, the mRNA analogues cross-linked preferentially to the 40S subunit, to the proteins and to a lesser extent to the 18S rRNA. Cross-linked nucleotides of 18S rRNA were identified previously. In the present study, it is shown that among the proteins the main target for cross-linking with all the mRNA analogues tested was protein S3 (homologous to prokaryotic S3, S3p); minor cross-linking to protein S2 (S5p) was also detected. Both proteins cross-linked to mRNA analogues in the ternary complexes as well as in the binary complexes (without tRNA). In the ternary complexes protein S15 (S19p) also cross-linked, the yield of the cross-link decreased significantly when the modified nucleotide moved from position +5 to position +12 with respect to the first nucleotide of the P site bound codon. In several ternary complexes minor cross-linking to protein S30 was likewise detected. The results of this study indicate that S3 is a key protein at the mRNA binding site neighboring mRNA downstream of the codon at the decoding site in the human ribosome.

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.

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.