Dmitri
Graifer
ab and
Galina
Karpova
*a
aLaboratory of Ribosome Structure and Functions, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia. E-mail: karpova@niboch.nsc.ru
bDepartment of Molecular Biology, Novosibirsk State University, Novosibirsk, 630090, Russia
First published on 13th November 2012
Ribonucleic acids (RNAs) are biopolymers that play key roles in many processes crucial for the life of cells. Functions of RNAs are generally mediated by their specific interactions with proteins and ribonucleoproteins (supramolecular RNA-protein complexes) that take place within complexes of RNA ligands with proteins or ribonucleoproteins. These interactions are defined by the molecular environment of the RNA ligand in the complex. A beneficial approach for studying such complexes involves site-directed cross-linking: RNA derivatives that contain chemically reactive groups form covalent bonds (cross-links) with those structural elements of the protein or ribonucleoprotein that neighbor the RNA ligand. Such RNA derivatives have been successfully used to study the interaction of messenger RNA (mRNA) with the protein synthesis machinery. By applying this approach to studies of the protein translation machinery of higher organisms, chemically reactive RNA derivatives have provided unique information that could not have been obtained by other approaches to date. This article presents a brief review of the types of RNA derivatives used as mRNA analogs, and their structures and synthesis, together with methods for identification of cross-linking targets. Furthermore, we summarize current data on mRNA interactions occurring during the course of protein synthesis in higher organisms.
![]() Dmitri Graifer | Dmitri Graifer is a leading scientist at the laboratory of Ribosome Structure and Functions headed by Prof. Galina Karpova, at the Novosibirsk Institute of Chemical Biology and Fundamental Medicine SB RAS. He obtained his Ph.D. degree in 1985 at the Novosibirsk Institute of Organic Chemistry, Biochemical Dept. In 1999 he was awarded the Russian State Prize in fields of science and technique. Since 2004 he has been an Associate Professor, and in 2008 he obtained the degree of Doctor of Chemical Sciences. The main topic of D. Graifer's research concerns studying structural and functional organization of human ribosomes with the use of site-directed cross-linking. |
![]() Galina Karpova | Galina Karpova obtained her Ph.D. degree in 1976 at the Novosibirsk Institute of Organic Chemistry, Biochemical Dept. Since 1980, she has headed the group (and the lab since 1990) of Ribosome Structure and Functions at the Novosibirsk Institute of Bioorganic Chemistry SB RAS (since 2003 Novosibirsk Institute of Chemical Biology and Fundamental Medicine). In 1990 she obtained the degree of Doctor of Chemical Sciences, and since 1993 she has been a Professor. In 1999 she was awarded the Russian State Prize in fields of science and technique. The main research field of G. Karpova's lab during the last 20 years has concerned studying structural and functional organization of human translational machinery. |
Various approaches have been used to study cellular processes involving RNAs, the protein synthesis apparatus being the subject of the majority of studies. As early as the 1970s, such studies on the interaction of aminoacyl-tRNA1 and mRNA2 with the ribosome were pioneered by D.G. Knorre with the use of site-directed cross-linking (affinity labeling). This approach is based on chemically active analogs of the ribosomal RNA ligands, which contain reactive groups that cross-link to ribosomal components in contact with or within a close proximity to the ligand. The affinity labeling is performed with mRNA and/or tRNA analogs bound within ribosomal complexes that imitate defined steps of translation, which is possible if the cross-linker does not interfere with the formation of such complexes. After the formation of cross-links, the identity of the covalently bound ribosomal components can be determined, and conclusions drawn concerning the structure of the ribosomal ligand binding centers.
Systematic studies of bacterial ribosomes were performed in the 1980s with the use of tRNA derivatives containing aromatic azides scattered throughout the molecule3 and mRNA derivatives consisting of short (3–12 nucleotides) oligoribonucleotides bearing a cross-linker (generally an alkylating 2-chloroethyl arylamine moiety) at the 3′ or 5′-end.4 These studies revealed for the first time ribosomal proteins located in tRNA binding sites3 and the mRNA binding center4 of the bacterial ribosome. Subsequently, the ribosomal mRNA binding center was studied by various groups using long (30–50 nucleotides) synthetic mRNA analogs bearing photoactivatable 4-thiouridine residues (e.g., see ref. 5–8). These cross-linking studies enabled the X-ray crystallographic data that followed to be correctly interpreted in order to reveal the structure of the bacterial ribosome at the atomic level (for a review, see ref. 9,10) and to make fundamental conclusions regarding the molecular mechanisms of translation.
Despite their general similarity to bacterial ribosomes, ribosomes from higher organisms (particularly human) are much more complicated and less well studied. This can be explained, in part, by the fact that several approaches used with success for studying bacterial ribosomes are not applicable yet to mammalian ribosomes. For example, approaches based on the reconstitution of active ribosomes from total ribosomal protein and rRNA are not possible with ribosomes of higher organisms. Furthermore, X-ray crystallography has only been successfully applied to lower eukaryotic ribosomes free from mRNA and tRNAs.11–13 This is why RNA analogs bearing cross-linkers at specific locations are the most suitable tools currently available for interrogating the interactions of RNA ligands with the mammalian translational machinery.
Here, we give an overview of the protein synthesis apparatus and describe current approaches for the synthesis of RNA analogs used for studying the mammalian translation system. These RNA analogs include synthetic mRNAs containing 4-thiouridines and derivatives of short oligoribonucleotides that bear photoactivatable perfluorophenyl azide cross-linkers at specific locations. We also describe an original method for site-specific introduction of a cross-linker into long, highly structured RNAs, and their application for studying the human translation system using the example of the hepatitis C virus internal ribosome entry site (IRES). Finally, we review the use of site-directed cross-linking in advancing our understanding of the fine structure of the ribosomal mRNA binding site, the arrangement of the hepatitis C virus IRES binding site on the small ribosomal subunit, and the molecular basis for recognition of mRNA stop codons by the translational termination factor eRF1.
Each tRNA is specific to only one aa; the correspondence of mRNA codons to aa residues is governed by a genetic code that is practically universal for cytoplasmic ribosomes from all kingdoms of life. Translation is terminated when one of three stop codons (UAA, UAG or UGA) falls at the A site. These codons are recognized by specialised ‘class 1’ termination factors (in eukaryotes, eRF1) that trigger hydrolysis of the complex ester bond between the polypeptide and the final tRNA, thus releasing the newly synthesized protein from the ribosome.
The ribosome is a highly complex molecular machine consisting of two subunits, one small and one large, each of which contains rRNA and several dozen ribosomal proteins. The structures of ribosomes from bacteria and eukaryotes share significant similarity. In particular, the secondary structures of their respective rRNAs have a universally conserved core. Furthermore, approximately 2/3 of ribosomal proteins in eukaryotic ribosomes have a bacterial homologue. However, eukaryotic rRNAs are longer than their bacterial counterparts and contain additional fragments that do not belong to the conserved core, and eukaryotic ribosomal proteins are generally larger than their bacterial homologues. Eukaryotic ribosomal subunits also contain additional proteins (about 1/3 of the total number) that have no homologues among bacterial ribosomal proteins.
The small ribosomal subunit (30S in prokaryotes and 40S in eukaryotes) contains an mRNA binding centre, including the decoding site where an aminoacyl-tRNA is selected that is cognate to the A-site mRNA codon. The large subunit contains the peptidyl transferase center responsible for catalysis of peptide bond formation (Fig. 1). Three tRNA binding sites, namely A, P and E (E is the site at which the discharged tRNA binds before it exits the ribosome), are formed by the interfacial surfaces of both subunits. During translation, the ribosomal mRNA binding center not only interacts with mRNA codons and tRNAs at the A and the P sites, but also binds a 30–50 nucleotide mRNA fragment that contains these codons.14,15 The most common approach used to obtain model complexes for studying the interaction of mRNA and tRNAs with the ribosome has been the direct binding of the ligands to the ribosome in the absence of translation factors and at Mg2+concentrations of 10–20 mM (which are approximately one order of magnitude higher than that in the cell). Under these conditions, stable complexes are formed in which the tRNA is bound to the P site (for which it has the highest affinity relative to the A and E sites), where it interacts with a cognate mRNA codon.16–18 Application of short oligoribonucleotide derivatives (up to 12 nucleotides long) as mRNA analogs is advantageous for the successful study of mammalian ribosomes, since they have very low intrinsic affinity for the ribosome, and their binding is hardly detectable in the absence of tRNA.19–22 However, in the presence of a tRNA cognate to one of the mRNA codons, the analog becomes tightly bound to the ribosome and is fixed in a defined position by interaction with the tRNA anticodon at the P site.19–22 Thus, the position of the derivatized mRNA analog nucleotide in the ribosome can be unambiguously governed by a tRNA.
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Fig. 1 Simplified scheme of the protein synthesis on the ribosome. The tRNA carrying the nascent peptide chain is at the P site, and the mRNA codon for the next amino acid (aa) residue to be incorporated is at the A site, where it is recognized by the anticodon of tRNA carrying the respective aa. When the ribosome performs the catalysis of the peptide bond formation, the peptide chain elongated by one aa residue becomes attached to the A site tRNA, and the tRNA at the P site is deacylated. Then the ribosome moves towards the 3′ end of the mRNA, so that the peptidyl-tRNA, together with the respective mRNA codon, moves from the A to the P site. The deacylated tRNA moves from the P to the E site, at which it can leave the ribosome, and the A site programmed with the next mRNA codon becomes vacant for the new, incoming aa-tRNA. |
Although eukaryotic ribosomes are also able to translate viral RNAs, the mechanisms of the translation initiation are generally different to cellular RNAs. Cellular mRNAs contain a special 7-methylguanosine cap structure at their 5′-terminus that is recognized by the initiation factor eIF4E (“e” refers to “eukaryotic”), which, together with other initiation factors, promotes the binding of 40S subunits in complex with the initiator Met-tRNAiMet. Subsequently, the small ribosomal subunit ‘scans’ along the mRNA until it is positioned at the first AUG codon at the P site, where the first codon interacts with the initiator tRNAiMet (for a review, see ref. 23): This is considered the key event of translational initiation. An alternative mechanism is utilized by certain uncapped viral RNAs that contain highly-structured fragments in their 5′-untranslated regions (upstream of the start AUG codon), called IRESs (internal ribosome entry sites), that positions the 40S subunit on the mRNA in a manner that directly places the AUG codon in the P site region (reviewed in ref. 24). In particular, IRES-mediated translation initiation is inherent to the RNA of the hepatitis C virus (HCV), one of the most dangerous human pathogens.
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Fig. 2 Basic scheme of studying the molecular environment of mRNA in the ribosome by site-directed cross-linking with the use of mRNA analogs bearing photoactivatable groups at the designed locations. 1D PAGE and 2D PAGE indicate one-dimensional and two-dimensional polyacrylamide gel electrophoresis, respectively. |
Identification of small subunit rRNA nucleotides cross-linked to labeled mRNA analogs is performed as follows (Fig. 3). rRNA is easily isolated from the modified ribosomes (or small subunits if the ribosomes were separated into subunits after cross-linking) by a standard procedure including extraction of proteins into water-saturated phenol with subsequent ethanol precipitation of the RNA. Regions of rRNA containing cross-linked mRNA analogs are then identified by hydrolysing the rRNA with RNase H in the presence of DNA oligomers that are complementary to specific rRNA sequences,21,28,29 thereby enabling rRNA cleavage at the designed sites. The resulting RNA fragments are separated by denaturing polyacrylamide gel electrophoresis (PAGE). Staining of the gels makes it possible to visualize all the RNA fragments, which can then be easily identified as their lengths correspond to the rRNA sites chosen for the RNase H cleavage; autoradiography of the gels shows which fragment contains the label. Identification of the cross-linked rRNA nucleotide is performed by reverse transcription using the modified rRNA as a template,30 and taking into account that as reverse transcriptase synthesizes DNA copies from the RNA template, it stops or pauses at the cross-linked RNA nucleotide. Primers for the reverse transcription (typically deoxy-20-mers), are designed based on the results of the RNase H experiments. The cross-linking site is generally assumed to be the nucleotide at 5′ to the primer extension end (pause) site, the position of which is determined by PAGE analysis of the primer extension products in parallel with the sequencing of unmodified rRNA, using each of the dideoxynucleoside triphosphates. This analysis allows identification of the exact sequence of the DNA transcript synthesized on the cross-linked rRNA and, consequently, the position of the cross-linked nucleotide in the rRNA. Control experiments are carried out with the rRNA isolated from unmodified subunits and processed in an identical manner, but in the absence of chemically reactive ribosomal ligands.
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Fig. 3 Principal steps of identification of cross-linked nucleotides of the target RNA. |
Identification of the ribosomal proteins cross-linked to mRNA analogs is more complicated than that of the rRNA nucleotides, with the choice of methodology depending on the identity of the cross-linked protein.22,25,31 To identify cross-linked proteins, modified ribosomes are first dissociated into subunits by centrifugation in a sucrose density gradient, before isolating total ribosomal protein from small subunits. In studies using short mRNA analogs, labeled proteins were identified using one- and two-dimensional PAGE in a variety of electrophoresis systems.32 Staining of the gels made it possible to visualize all unmodified proteins based on their known electrophoretic position, with autoradiography, then identifying the locations of the cross-linked proteins. Although the locations of radioactive spots did not exactly correspond to those of the respective unmodified proteins (due to the retarding effect of the cross-linked oligoribonucleotide on electrophoretic mobility), it has been possible to identify cross-linked proteins by comparing mobilities in different electrophoretic systems.22,25 In some cases, the identity of cross-linked ribosomal proteins has been confirmed with the use of antibodies against specific mammalian ribosomal proteins,22 mass spectrometry27 and other approaches (e.g., ref. 22). An example illustrating the identification of the ribosomal protein S26e (rpS26e) cross-linked to mRNA analogs is presented in Fig. 4.
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Fig. 4 Identification of the rpS26e cross-linked to mRNA analogs whose ATB-derivatized uridine (U*) was placed in position −4, −6 or −9 (the respective mRNA analogs could be found in Fig. 10). (A) Analysis of the proteins cross-linked to one of the mRNA analogs by a two-dimensional PAGE after hydrolysis of the cross-linked RNA with RNase A. The cross-linked protein is marked with an asterisk (*). Spots of several unmodified proteins in the stained gel are indicated according to ref. 32. The location of the radioactive spot is indicated by the dotted line. (B) Identification of the cross-linked rpS26e protein by immunoblotting after 1D PAGE separation of 40S proteins untreated with RNase. Lanes 1, 2 and 3 correspond to the results with the complexes where U* was in the position −4, −6 or −9, respectively. Lane K corresponds to a control experiment with proteins isolated from unmodified 40S subunits. Panel (a) is an autoradiogram, panel (b) presents the identification of rpS26e with specific antibodies. Note that only a minor extent of the ribosomal rpS26e underwent the cross-linking, so the major part of it remained unmodified; so, immunoblotting developed strong bands of the unmodified rpS26e, and weaker bands of the protein cross-linked to mRNA analogs. |
With 30–50 nucleotide long mRNA analogs, direct identification of the cross-linked proteins by PAGE is highly ambiguous since the effect of the cross-linked RNA on protein mobilities is too strong. In these cases, two alternative approaches have been applied to identify the cross-linked proteins. The first utilized mRNA analogs carrying a single internal 32P label in the 3′-phosphate of the nucleotide bearing the cross-linker. This approach enabled hydrolysis of the cross-linked RNA without loss of the label followed by PAGE analysis, since modified proteins were only cross-linked with mono- or dinucleotide residues.27 This approach was used to identify amino acids of the translational termination factor eRF1 that cross-linked with the first nucleotide of the A site stop codon in an mRNA analog.26 The second approach is based on the application of uniformly labeled mRNA analogs. This allows the removal of the majority of RNA that is cross-linked using RNase, leaving a very short labeled oligonucleotide fragment.33 Both approaches have their specific disadvantages. Preparation of RNAs containing a single internal label in amounts sufficient for cross-linking experiments is sometimes problematic because of the low yield of a product obtained as a result of ligation of the two “halves” (see above). When using uniformly labeled RNAs, >95% of the label is lost in the course of the exhaustive RNase hydrolysis, which requires large amounts of labeled RNAs and other components that may be not easily available.
Site-directed cross-linking allows not only identification of the cross-linked proteins, but even the specific peptides that have been modified. A special methodology for the identification of cross-linked peptides of proteins was initially suggested,26 and then developed and successfully applied.34–37 The approach is based on the specific cleavage of the cross-linked protein with various proteolytic agents and subsequent separation of the resulting labeled and modified peptides by PAGE. These peptides can be identified by comparing their electrophoretic mobilities with those predicted from the known cleavage specificity of the proteolytic agent used. In the example given on the study of the termination factor eRF1, mutant forms of the factor were also used that contained single or double amino acid substitutions in the regions of interest that allowed fine mapping of protein fragments containing cross-links. These substitutions either provided new artificial methionine residues or eliminated natural methionines in the factor (methionine is a target for specific cleavage with cyanogen bromide).26,35,36
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Fig. 5 Alkylating aromatic 2-chloroethylamine groups introduced at the terminal phosphate or ribose of oligoribonucleotides. |
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Fig. 6 Structure of 4-thiouridine (s4U) (A) and a complex of the ribosome with a purine-rich s4U-containing mRNA analog applied for studying the ribosomal decoding site and the stop codon recognition site on the eRF1, (B). The mRNA analog is positioned on the ribosome by its GAC triplet that interacts with the cognate tRNAAsp at the P site. The subsequently modified stop codon s4UGA is at the A site where it can interact with the eRF1. The radioactive labeled phosphate is between the s4U and G of the stop codon, which makes hydrolysis of the cross-linked mRNA analog possible, without the loss of the label. |
s4U-containing mRNA analogs were extensively used in studies of bacterial ribosomes by groups led by R. Brimacombe and A. Bogdanov (e.g., see ref. 5,6), and P.Wollenzien.7,8 Initial reports on similar studies of human ribosomes30,41 revealed that it was difficult to achieve the unambiguous position of mRNA (and therefore, its s4U residues) on the ribosome by tRNA cognate to one of mRNA codons targeted to the P site. The problem was related to the conditions used for the in vitro assembly of the model ribosomal complexes, i.e. the absence of translation factors, and incubation with 10 mM of Mg2+ (which is 5-fold over that in the cell) at room temperature. Under these conditions the mRNA analogs had a large intrinsic affinity for the ribosomes in the absence of tRNA, which allowed them to bind the ribosome at ambiguous positions. Since the binding level and cross-linking pattern of mRNA analogs did not depend on the presence of tRNA, this implied that tRNA caused no significant change in the location of the mRNA on the ribosome.30,41 To overcome these difficulties, special purine-rich mRNA analogs were designed whose intrinsic affinities to 80S ribosomes were relatively low (an example is presented in Fig. 6B).27,42,43 Using these analogs, addition of tRNA cognate to the selected codon resulted in an increase in the level of mRNA binding, and the appearance of new cross-links that could be unambiguously assigned to complexes containing an mRNA analog fixed to the ribosome as a result of a codon–anticodon interaction of tRNA and mRNA at the P site (Fig. 6B). It should be noted that the problem concerning ambiguous binding of long mRNA analogs to the ribosome only arose under conditions of supraphysiological Mg2+ concentrations, but not more physiological levels in the presence of translation factors.33,44 However, in the majority of studies, model complexes were not assembled under these more physiological conditions, presumably because the respective mammalian translation factors were difficult to obtain at this time.26,27,30,41–43 Thus, a disadvantage of the relatively long s4U-containing mRNA analogs was that only special adenine-rich derivatives were suitable under the described ‘factor-free’ conditions, which in turn significantly restricts the sequence possibilities of the mRNA. Finally, a method for preparation of mRNA analogs containing a single 32P 3′ to the s4U residue (which is most suitable for precise identification of cross-linking products) often gives low yields of the final RNA that are insufficient for identification of the cross-linked products.
It is worth mentioning here that approaches based on enzymatic incorporation of photoactivatable nucleotides into RNA are also used to study RNA–protein interactions that do not directly relate to the translational machinery. So, an RNA containing 8-azido-adenosine residues obtained by T7 transcription from the appropriate DNA template was used in a cross-linking study with the coat protein of the Escherichia coli phage MS2.45 Besides, a method utilizing in vivo incorporation of s4U into RNAs by incubation of the living cells with s4U with subsequent UV-irradiation was elaborated and applied for studying RNA–protein interactions between s4U-containing RNA transcripts and RNA-binding proteins (RBPs) or microRNA-containing ribonucleoprotein complexes (RNPs) that regulate maturation, stability, transport, editing and translation of these transcripts.46 This method called PAR CLIP (photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation) is based on the immunoprecipitation of the cross-linked protein, the conversion of the cross-linked RNA fragments into a cDNA library and its sequencing; cross-linking sites are revealed by thymidine to cytidine transitions in the cDNAs. Photoactivatable nucleosides other than s4U (6-thioguanosine, 5-bromouridine and 5-iodouridine) are not good for PAR CLIP, since they provide too low yield of RNA–protein cross-links.46 The use of PAR CLIP with s4U allowed the authors to directly identify transcriptome-wide RNA sites for binding of several regulatory RBPs and RNPs in live cells.
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Fig. 7 Structures of the ATB-modified nucleosides and a terminal phosphate, which were present in short mRNA analogs, derivatives of oligoribonucleotides and the formation of biradical nitrene upon UV-irradiation of the ATB group. |
A condensation reaction with aliphatic diamines (ethylene diamine or isopropylene diamine) is used to introduce an amino-spacer at the terminal phosphate of an oligoribonucleotide, with the help of a basic catalyst (N-methyl imidazole or dimethylaminopyridine) and a combination of triphenylphosphine with 2,2′-dipiridyl disulfide as a condensing agent (e.g., see ref. 19). In order to obtain an oligoribonucleotide derivative bearing an amino spacer at the N7 atom of the guanine, alkylation with 4-(N-2-chloroethyl-N-methylamino)benzylamine is employed under specific conditions that practically exclude the modification of other nucleotides.29 Evidently, this approach can only be applied to oligoribonucleotides that contain a single guanosine residue at a defined position. Lastly, the introduction of an amino spacer into a selected uridine or adenine is performed during the solid phase chemical synthesis of the oligoribonucleotide. 5-Br-uridine or 8-Br-adenosine monomers that contain all the groups necessary for oligoribonucleotide synthesis are used at the appropriate step in order to incorporate the modified nucleotide at the specified position.51 Following synthesis, the Br within the oligomer is replaced by ethylene diamine or isopropylene diamine residues using a nucleophilic substitution reaction performed with the respective diamine.51
When analyzing cross-linking results obtained using these ATB-derivatized RNA ligands, one should take into account that the maximum distance separating the derivatized RNA nucleotide and the reactive nitrogen of the cross-linker could be up to 11–14 angstroms, depending on the spacer structure. Therefore, the cross-linked ribosomal components could be those that either make direct contacts with the RNA ligand, or those that are located in close vicinity of, but not in direct contact with, the RNA.
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Fig. 8 Scheme of the site-specific introduction of a photoactivatable group into a selected RNA site, based on the complementary-addressed alkylation of the RNA with [4-(N-2-chloroethyl-N-methylamino)benzyl]-phosphoramides of oligodeoxyribonucleotides. |
The approach outlined above was successfully used to introduce a cross-linker in defined positions of the HCV IRES, which could be considered as a peculiar mRNA analog consisting of nucleotides 40–372 in the genomic RNA of HCV.56 To introduce a cross-linker into the IRES, the respective 32P-labeled RNA fragment was obtained by transcription with T7 RNA polymerase from a specially synthesized DNA template,57 an approach similar to that used for preparation of the 30–50 nucleotides long mRNA analogs described above. RNA containing biotin residues were obtained by transcription with biotinylated nucleoside triphosphates in several studies. Application of biotinylated RNAs made it possible to isolate cross-linked products containing the IRES derivatives by affinity chromatography on avidin-agarose.54 The RNA sites used to introduce the cross-linker were chosen based on available data regarding the domains and subdomains of HCV IRES involved in the binding to the small ribosomal subunit. To increase the yield of the IRES alkylation with deoxyoligomer derivatives, “helper oligomers” were employed that promoted the disruption of the IRES secondary structure, thereby allowing binding of oligomers containing the alkylating group to the structured RNA. The set of HCV IRES derivatives that were obtained by this approach54,55 (Fig. 9) helped to reveal ribosomal components that take part in HCV IRES binding to the human small ribosomal subunit that plays a key role in the initiation of the viral RNA translation (reviewed in ref. 24).
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Fig. 9 The secondary structure of the HCV IRES24 (the initiation AUG codon is underlined). RNA sequences complementary to the deoxy-oligomers used for site-specific modification of the HCV IRES used in ref. 54 (A) and in ref. 55 (B) are shown with thick lines with arrows indicating the 5'-phosphates derivatized with alkylating groups. RNA sequences complementary to “helper oligomers” used together with the alkylating derivatives are marked with dotted lines. Nucleotides of the HCV IRES modified with the oligonucleotide derivatives are shaded. |
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Fig. 10 ATB-derivatized mRNA analogs applied for studying human translational machinery. The derivatized nucleotides or phosphates are in red and marked with asterisks. mRNA codons targeted to the ribosomal P site to fix the mRNA at the designed position on the ribosome are in blue. Position +1 conventionally corresponds to the first nucleotide of the P site codon. Positions of mRNA nucleotides upstream and downstream of this nucleotide are marked “−“ and “+”, respectively. |
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Fig. 11 Summary of the structural elements of the human ribosome that neighbor particular positions of mRNA, according to cross-linking data with ATB-derivatized mRNA analogs. mRNA is shown as diagrams; positions of the mRNA nucleotides on the ribosome are indicated and the codons interacting with tRNAs are marked with colored lines. Upper diagram shows the protein environment of mRNA (the main targets of cross-linking are in bold).22,25,31,34,37 The bacterial counterparts are shown in brackets. Below, the mRNA–rRNA neighborhood21,22,28,29,59 is presented on the secondary structure of the human rRNA from the small ribosomal subunit taken from www.rna.ccbb.utexas.edu. |
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Fig. 12 Components of mammalian translational machinery contacting mRNA during the translation initiation.33,44 For details, see the Fig. 11 legend. eIFs are subunits of the initiation factors. |
Comparison of the early data obtained from the application of short ATB-derivatized mRNA analogs (Fig. 11) with later data using longer synthetic s4U-containing mRNAs (Fig. 12) indicates that both tools gave similar results, despite the model complexes being formed in different ways. Discrepancies between the assigned environments of particular mRNA positions observed with these different mRNA analogs are expected, however, mainly due to differences in the chemical and stereochemical properties of the cross-linkers (s4U and ATB-modified nucleotides). Some dissimilarity in the cross-linking patterns obtained with these mRNA analogs could also be due to differences in how the mRNA analogs form their respective complexes. Interestingly, the direct interaction of the translation initiation factor (eIF) subunits with mRNA was also identified using this approach.33 These data, together with the finding that a eukaryote-specific motif in rpS26e cross-links to derivatized mRNA nucleotides in the same positions as those contacting eIF3, led to new ideas regarding how mRNA is recruited to the small ribosomal subunit during translation initiation in eukaryotes.37
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Fig. 13 Spatial structure of the human translation termination factor eRF1 according to ref. 69 (right) and a magnified view of its N-domain (left), where conserved amino acid motifs are shown that were found to cross-link to derivatized U, A or G in the A site bound stop codon of mRNA analogs within the ribosomal termination complexes.26,35,36 |
To examine the position of the mRNA stop codon purines relative to eRF1 in translation termination complexes, short mRNA analogs were used that bore an ATB-derivatized guanine35 or adenine36 in a stop codon targeted to the A site. As with the s4U-containing mRNA analog described above, cross-linking to eRF1 was specific for those analogs containing the ATB-derivatized nucleotides in a stop codon, since it did not occur when control mRNA analogs were used that bore the derivatized A or G within a sense codon.35,36 Surprisingly, these experiments indicated that different evolutionary conserved motifs of the eRF1 N-domain cross-linked to modified guanines and adenines (Fig. 13). These results were then used to guide molecular modeling of eRF1 in the human ribosomal termination complex, using available structural data and software. This indicated that the peptide environments of the stop codon guanines and adenines are compatible with two alternative conformations of the eRF1 N-domain in the mammalian ribosomal termination complex. The capacity of the ribosome-bound eRF1 to adopt alternative conformations was suggested to be essential for recognition of all stop signals by a single termination factor.36 This recognition mode is principally different from that in bacteria, where two termination factors, RF1 and RF2, decode UAA/UAG and UAA/UGA, respectively. Thus, data obtained with the ATB-derivatized short mRNA analogs provided new insights into the mechanism of the stop codon decoding in the course of translation, and are in an agreement with NMR studies that also suggested rearrangement of the eRF1 N-domain during stop codon recognition.63
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Fig. 14 Structural model of the mammalian small ribosomal subunit obtained from cryo-electron microscopy data (adapted from ref. 65, Protein Data Bank accession number 2ZKQ, a view from the solvent side). Locations of ribosomal proteins cross-linked to HCV IRES nucleotides are shown in various colors (except for rpS3a, whose location became known from the later X-ray study,11 where this protein is designated as rpS1e, shown by the dotted line). All other proteins that have bacterial counterparts are shown in green, and proteins specific to eukaryotes are not shown. The gray balls represent the 18S rRNA. |
Footnote |
† This review article is dedicated to the memory of Har Gobind Khorana, a scientist whose contribution to understanding the functions of nucleic acids is invaluable. |
This journal is © The Royal Society of Chemistry 2013 |