*Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poland;
and
Institute of Biochemistry of Free University, Berlin, Germany
Abstract
The structure-function relationship in RNA molecules is a key to understanding of the expression of genetic information. Various types of RNA play crucial roles at almost every step of protein biosynthesis. In recent years, it has been shown that one of the most important structural elements in RNA is a wobble pair G-U. In this paper, we present for the first time an analysis of the distribution of G-U pairs in eukaryotic 5S ribosomal RNAs. Interestingly, the G-U pair in 5S rRNA species is predominantly found in two intrahelical regions of the stems I and V and at the junction of helix IV and loop A. The distribution of G-U pairs and the nature of adjacent bases suggests their possible role as a recognition site in interactions with other components of protein biosynthesis machinery.
Introduction
In recent years, there has been much interest in understanding the structure-function relationships of RNA molecules in cellular processes. It is generally accepted that RNA molecules, apart from their functions as carriers of genetic information and structural scaffolds for proteins in various ribonucleoprotein complexes, also possess catalytic activity (Bartel and Unrau 1999
). A detailed understanding of the function of RNAs requires a precise knowledge of the rules governing formation of their higher-order structures.
From the very beginning of the study of RNA molecules, it has been known that assembly of higher-order structures of RNA is due not only to formation of the Watson-Crick base pairs (A-U and G-C), but also to a wobble pair (G-U). Later, it was found that the G-U base pair cannot be considered merely a simple substitute for the canonical base pairs. Various structural studies of RNA (NMR, X-ray diffraction) have shown that the G-U base pair causes some distortions in the helical regions of RNA (White et al. 1992
; Wohnert et al. 1999
). This is due to displacement of guanine residue toward a minor groove of the RNA helix relative to its location in the G-C pair, which results in a considerable loss of stacking 5' of guanosine (Mizuno and Sundaralingam 1978
). The consequence of such a structure is a lower helix stability (Freier et al. 1986
), which is further confirmed by the observation that loops most often occur on the 5' side of the guanine residue of the G-U base pair (Gautheret, Konings, and Gutell 1995
).
There are also several other examples which suggest that the distribution of the G-U base pairs in various RNAs is not totally random and is not directed only by structural constraints: they constitute highly specific recognition sites for proteins or other RNAs (McClain et al. 1999
; Reyes et al. 1999
). In the group I introns, a conserved G-U pair at the splice site participates in the formation of a catalytic core by binding other parts of the molecule (Reyes et al. 1999
; Strobel and Cech 1995
). It has also been shown that the G3-U70 pair located in the acceptor stem of tRNAAla from Escherichia coli contributes significantly to the specificity of interaction with its cognate aminoacyl-tRNA synthetase (McClain et al. 1999
; Hou and Schimmel 1988
).
Until now, very little has been known about the detailed function of ribosomal RNAs (Correll et al. 1997
). They were originally recognized as a scaffold for ribosomal proteins (Mueller et al. 1995
). The discovery of catalytic activity of certain RNAs allows speculation that they may take an active part in the process of protein biosynthesis as well (Dai, De Mesmaeker, and Joyce 1995
; Porse and Garrett 1999
).
Recently, an extended analysis of the distribution of G-U pairs in the large ribosomal RNAs, with emphasis on their possible function, was published (Gautheret, Konings, and Gutell 1995
). The smallest RNA component of almost all ribosomes is 5S ribosomal RNA. The size of this RNA, combined with the availability of relatively fast RNA sequencing methods, resulted in accumulation of a large number of data concerning this molecule (Barciszewska, Erdmann, and Barciszewski 1996
). In this paper, we analyze the distribution of the G-U base pair in the secondary structure of eukaryotic 5S rRNAs. This extensive analysis provides some new information about possible functional roles of G-U pairs in 5S rRNA with respect to protein and/or RNA binding as well as potential catalytic function.
Materials and Methods
5S rRNA nucleotide sequences analyzed in this paper were taken from our data banks (Szymaski et al. 1997, 2000
). The database entries use the format of the EMBL Nucleotide Sequence Data Bank. The 5S rDNA nucleotide entries contain the 5S rRNA coding sequence, as well as information on the length of the original clone and the location of the structural gene.
Files with the primary-structure data and the nucleotide sequence alignments are available via the World Wide Web at http://rose.man.poznan.pl/5Sdata/index.html. Any nucleotide sequence can be retrieved with the taxonomy browser or from an alphabetical list of organisms (Szymaski et al. 2000
).
Nucleotides occurring at all positions in 285 eukaryotic 5S rRNAs were taken into account. The frequency and ratio (%) of G-U pairs found were calculated.
Results and Discussion
The prerequisite for an understanding structure-function relationship in nucleic acids is data on their primary structure. It is generally accepted that the longer DNA or RNA chains provide much more complete data. However, for large molecules of ribonucleic acids, it is difficult to perform detailed analysis of the secondary-structure features using either computer methods or experimental approaches (Barciszewska, Erdmann, and Barciszewski 1994
; Szyma
ski et al. 1997
). On the other hand, small RNA species contain very little information that may be useful for phylogenetic analyses, but they constitute very good models for structural studies. 5S ribosomal RNA, despite its relatively small size of 120 nt and molecular weight of 40 kDa, is one of the best RNA molecules to be studied (Barciszewska, Erdmann, and Barciszewski 1996
). More than 30 years of research on 5S rRNA has resulted in several models of its secondary structure. However, very little is known about the spatial organization and tertiary-structure elements of this molecule (Barciszewska, Erdmann, and Barciszewski 1996
).
In this paper, we analyzed the distribution of the G-U base pair in 285 5S ribosomal RNA sequences isolated from eukaryotes (Szymaski et al. 2000
). If one considers the size of 5S rRNA, it seems that this molecule is very suitable for analysis of the influence of a nonstandard base-pairing on the structure and function of RNA. First, we analyzed sites (positions) in the secondary structure of eukaryotic 5S rRNAs at which the G-U pair occurs (fig. 1
). Although the G-U pairs can be found at almost every position in the double-stranded regions of 5S rRNA molecule, there are some strong preferences with respect to localization as well as orientation at particular sites (fig. 1 ). Within the secondary structure of eukaryotic 5S rRNA, we clearly identified two regions occupied mostly by the G-U base pairs. One of these was located in stem I, close to loop A. G-U base pairs were at positions 7112 and 8111. They were present in 87% of all eukaryotic 5S rRNA sequences. The second site was found in stem V, where the G97-U79 base pair occurs in 81% of eukaryotic 5S rRNAs. From earlier studies, it is known that an effect of the wobble pair on the overall structure of double-stranded regions of RNA depends on the nature of the adjacent base pairs (Wohnert et al. 1999
). It seems that a destabilizing effect of the G-U pair is partly due to the loss of stacking 5' of guanine residue and can be compensated by the presence of purine at this position (Gautheret, Konings, and Gutell 1995
). Thus, we divided all G-U sites found in 5S rRNAs into two groups: compensated (with purine at the position 5' of G) and uncompensated (with pyrimidine at the position 5' of G) (Gautheret, Konings, and Gutell 1995
). Generally, our analysis showed only two intrahelical positions in which the G-U base pair is predominant (table 1
). Interestingly, over 70% of the 5S rRNA nucleotide sequences contain uncompensated G-U pairs in either or both of the two regions. The high frequency of occurrence of uncompensated G-U base pairs strongly suggests that this motif in the structure is the real recognition target for 5S rRNA interactions, for example, with L5 or TFIIIA (Theunissen, Rudt, and Pieler 1998
). Preliminary analysis of eubacterial 5S rRNA did not confirm this pattern. This is reasonable, because a network of interactions of prokaryotic 5S rRNA is different from its eukaryotic counterpart (unpublished data).
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Strikingly, we found a very strong preference for the wobble pair in stem V. In 27% of the analyzed 5S rRNA pairs, there is a U96-U80 pair 5' of the guanosine of the G97-U79 pair. This kind of arrangement is highly conserved and characteristic for 5S rRNAs from multicellular animals. Interestingly, the crystal structure of the synthetic RNA duplexes showed that the presence the U-U pair induces some changes in the overall geometry of the double helix (Baeyens, De Bondt, and Holbrook 1995
). There are also some cases in 5S rRNA in which the G97-U79 pair is replaced by U97-U79 (Dallas and Moore 1997
; Leonits and Westhof 1998). When this position is occupied by Watson-Crick pair, another wobble pair, C96-A80, can also be found. This is particularly interesting in light of recent NMR studies of 5S rRNA from E. coli. The analysis of NOE patterns observed for G79-U82 and A94-C97 base pairs suggested similar duplex conformations for both G-U and A-C base pairs (Grüne et al. 1996
). This observation leads to the conclusion that the wobble base pairs play an important role in changing the structure of the RNA duplex (Limmer et al. 1996
; Rawlings, Matt, and Huber 1996
). The change or distortion of the regular helix geometry may be a recognition site for another RNA or protein molecule (Reyes et al. 1999
). There are only a few 5S rRNA structures in which stem V is composed exclusively of Watson-Crick base pairs, and the observed conservation of a wobble configuration in this region suggests that stem V of the 5S rRNA molecule may constitute a recognition site for protein or RNA binding (Wohnert et al. 1999
).
In addition to the intrahelical G-U pairs in stems I and V, there are two other sites at the helix-loop junction where they occur. One of these is located at the end of stem IV, next to loop A, where the conserved G66-U109 pair is present in all eukaryotic 5S rRNA sequences. It appears that this base pair plays crucial role in the formation of the higher-order structure of loop A, which serves as a hinge region where helices I, II, and IV meet (Limmer et al. 1996
; Theunissen, Rudt, and Pieler 1998
). Analysis of the point mutants in the loop A has shown that this region is important for the recognition of 5S rRNA by transcription factor IIIA (Theunissen, Rudt, and Pieler 1996
; Rawlings, Matt, and Huber 1998
). Another site for frequently occurring G-U pairs is the stem I/loop A junction. The pair formed between G110 and U9 is present in 17% of all analyzed nucleotide sequences of 5S rRNA. In both cases in which the G-U pair occurs at the helix-loop junction, the loop is located 5' of a guanine residue, thus compensating loss of stacking caused by the wobble conformation. Similar observations have been made for the G-U pairs in large ribosomal RNAs (Gautheret, Konings, and Gutell 1995
).
An effect of the G-U pair on the helix structure in stem I of E. coli 5S rRNA was studied by NMR spectroscopy. It was shown that the overall A-RNA helix geometry is distorted by the presence of the G9-U111 (G8-U111 in eukaryotic 5S rRNA) base pair (White et al. 1992
). The wobble pair does not stack with the preceding C8-G112 pair, but with the next C10-G110 pair, which closes stem I at the junction with loop A. It has been suggested that in such an arrangement, the G-U pair is responsible for stabilization of the complicated three-helix junction at the expense of the stem. In contrast to the eubacteria, this position, when occupied by a G-U pair in the eukaryotic 5S rRNAs, occurs in compensated conformation. It is difficult to conclude whether the effect of such an arrangement would be the same in a uncompensated one (Dallas and Moore 1997
). The NMR study of the acceptor stem of alanyl-tRNA from E. coli has shown that the presence of the G3-U70 base pair causes irregularity of the helix by displacing C71. This results in the decreased stacking between C71 and U70 (Limmer et al. 1996
).
Up to now, there have been only three G-U pairs for which some functional role has been ascribed. One is the G3-U70 pair in tRNAAla, which is responsible for recognition by the alanyl-tRNA synthetase (Hou and Schimmel 1988
; McClain, Gabriel, and Schneider 1996
; McClain et al. 1999
), the second is the G-U pair at the splice site of group I introns (Strobel and Cech 1995
), and the third is G-U pair in the ribosomal protein L32 pre-mRNA from yeast, which is recognized by the L32 protein (White and Li 1996
; Reyes et al. 1999
). One should notice that these pairs occur in different structural contexts. In tRNAAla, distortion of the acceptor stem caused by the G-U pair is compensated by guanine residue 5' of G. On the other hand, the conserved G-U pair in self-splicing introns always occurs in an uncompensated conformation (Strobel and Cech 1995
). The conformation of the G-U pair in L32 pre-mRNA is also uncompensated (Strobel and Cech 1996
). We can assume, that the importance of the G-U base pair as a determinant of specificity of an aminoacylation reaction is governed by its specific recognition by the aminoacyl-tRNA synthetase. This is due to the presence of the free exocyclic amine of guanine residue. Similarly, the functional groups present in the minor groove at the splice site of group I introns have been postulated to play a role in formation of the active catalytic core of the ribozyme (Strobel and Cech 1996
).
From the above observations, it is clear that the G-U base pairs introduce local distortions into regular helix geometry which may constitute recognition sites for both RNA and protein binding. By analogy to the situation observed in the group I introns, where the G-U pair is involved in the interaction with other parts of the RNA forming the catalytic core (Strobel and Cech 1995
), one can suggest that the uncompensated G-U base pairs in 5S rRNA are bound by some fragments of ribosomal RNA. There are, however, only a few known examples of the RNA-RNA complexes from which to draw general conclusions. On the other hand, there is evidence that 5S rRNA may interact with large ribosomal RNA. It has been recently shown that loop IV (loop E in eukaryotic 5S rRNA) of eubacterial 5S rRNA contacts domains II and V of 23S rRNA (Dokudovskaya et al. 1996
).
It is evident that 5S rRNA, as a part of large nucleoprotein structure, must form multiple contacts with its other components. Moreover, it has been shown that 5S rRNA is part of the eukaryotic elongation factor EF-2binding site (Nygard and Nilsson 1987
). It is not known whether ribosomal RNAs play a catalytic role in protein biosynthesis. It is difficult to ascribe a specific function to particular components of such a large complex as a ribosome. There are, however, some clues indicating that RNA might take an active part in this process. A model proposed for the orientation of 5S rRNA in the 50S subunit of E. coli ribosomes localizes stem-loop IV (stem V, loop E) close to the peptidyl transferase center (Dontsova et al. 1994
). It is interesting to note that in stem IV of E. coli 5S rRNA, there is a G-U pair at the position corresponding to the conserved G97-U79 pair in eukaryotic 5S rRNA.
Acknowledgements
This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Bundesministerium fur Forschung und Technologie, the Fonds der Chemischen Industie E.V., and the Polish Committee for Scientific Research.
Footnotes
William Taylor Saitou, Reviewing Editor
1 Keywords: 5S ribosomal RNA
wobble pairs
RNA secondary structure
2 Address for correspondence and reprints: Jan Barciszewski, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 Pozna, Poland. E-mail: jbarcisz{at}ibch.poznan.pl
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