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REPORT |
1 Max Perutz Laboratories, Institute of Microbiology and Genetics, 1030 Vienna, Austria
2 Institute of Molecular Biology, Johns Hopkins University, Baltimore, Maryland 21205, USA
Reprint requests to: Katharina Semrad, Max F. Perutz Laboratories, Institute of Microbiology and Genetics, Dr. Bohrgasse 9/4, 1030 Vienna, Austria; e-mail: katharina.semrad{at}univie.ac.at; fax: ++43-1-4277-53099.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Keywords: RNA chaperone; ribosomal proteins; trans splicing; RNA folding; 50S subunit
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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; Wimberly et al. 2000; Harms et al. 2001). Many of the ribosomal proteins have unusual extended basic tails that appear to wind their way through ribosomal RNA, acting to cement together the final structure. Most ribosomal proteins also have a globular domain that extends over the surface of the subunit, most often in solvent-exposed regions. There are, however, notable exceptions where the ribosomal proteins are located in the interface region between the large and small subunits of the ribosome where they might play direct roles in functionally critical intersub-unit interactions like S12 and S13 (Culver et al. 1999; Cukras et al. 2003). In addition, while ribosomal proteins are largely absent from the peptidyl transfer and decoding regions, they are the principal components of the stalk region of the large ribosomal subunit that is directly involved in binding and interacting with translation factors like EF-Tu and EF-G (Mohr et al. 2002). Finally, a variety of ribosomal proteins are directly implicated in ribosome function based on documentation of their motions during discrete steps of translation (Gao et al. 2003; Valle et al. 2003).
We focus here on the potential role of ribosomal proteins as RNA chaperones to prevent the ribosome or other RNA molecules from stably occupying misfolded conformational states. Earlier work from Belfort and coworkers demonstrated that the small subunit ribosomal protein S12 from Escherichia coli has ATP-independent RNA annealing and RNA displacement activities in vitro (Coetzee et al. 1994). These activities are associated with RNA chaperones because their actions increase the fraction of correctly folded RNA by either impeding the formation of misfolded structures or by destabilizing folding intermediates (Herschlag 1995). In a separate work the relevance of the in vitro activity of ribosomal protein S12 could be extended to an in vivo context where S12 overexpression rescued a misfolded group I intron to promote catalysis (Clodi et al. 1999). In vivo it was further demonstrated that the RNA chaperone StpA destabilizes tertiary structural elements of a group I intron, again ultimately leading to correct folding and associated catalysis (Waldsich et al. 2002). While it is unclear whether the documented RNA chaperone activity of S12 is implemented by the ribosome for optimal function, it is clear that such activities might be useful to guarantee the dynamic flexibility of the ribosome during the translation cycle.
To extend the initial observation made with S12, we here focus on the purification of the ribosomal proteins from the large ribosomal subunit from E. coli and their evaluation in an in vitro RNA chaperone assay (Coetzee et al. 1994; Zhang et al. 1995). We found that nearly a third of the large ribosomal subunit proteins from E. coli display RNA chaperone activity.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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). The overexpressed proteins were purified under denaturing conditions (6 M urea) over a cation column (with the exception of ribosomal protein L4, which was purified over an anion column) and eluted with an increasing salt gradient (see Table 1 and Materials and Methods). Purification was repeated when significant RNase activity remained. In the case that a second purification was performed, the respective second buffer, in which the proteins were dialyzed prior to the second purification, is indicated in Table 1. In some cases the same buffer as used for the first purification was chosen; in other cases the pH of the buffer was slightly increased to assure higher purity of the eluted proteins. We next used the previously developed trans splicing assay to evaluate the RNA chaperone activity of the collection of purified ribosomal proteins. In this assay the pre-mRNA of the thymidylate synthase (td) gene containing a group I intron is split in two halves. The first transcript, H1, contains the 5' exon sequences (549 nt) and 131 nt of the intron and the second transcript, H2, contains the 3' part of the intron (147 nt) and parts of exon 2 (23 nt) (Fig. 1A). The two independent constructs were transcribed using 35S-
-UTP and were then incubated together. The group I intron trans-splicing reaction was initiated with the addition of 32P-GTP, so that the resulting spliced product is doubly labeled (internally with 35S--UTP and at the 5' end with 32P-GTP). Trans splicing was performed in the absence and the presence of the respective ribosomal protein. At 55°C the H1 and H2 RNAs effectively anneal and fold into a productive molecule while at 37°C the RNAs fail to assemble into an active conformation. The group I splicing reaction is sensitive to lower temperatures. The individual purified ribosomal proteins were added to a final concentration of 2 µM as was used recently for assessing the RNA chaperone activity of StpA (Mayer et al. 2002). Higher protein concentrations (3 µM, 4 µM, 6 µM) were tested for ribosomal protein L19 and did not show an increase in the observed chaperoning activity. Addition of an RNA chaperone at 55°C does not further stimulate trans splicing (data not shown). Only when trans splicing was already reduced due to lower temperatures (37°C) does the presence of an RNA chaperone stimulate trans splicing. Figure 1B shows a representative time course of trans splicing in the presence of ribosomal protein L19 where the reaction is five times more efficient with L19 at 37°C than without L19 at 55°C (positive control). Ribosomal proteins L1, L13, and L15, among others, further increased trans splicing at 37°C, whereas others (L9, L7/L12) did not stimulate trans splicing (Fig. 2). Figure 3 summarizes the relative trans splicing rates of proteins from the large ribosomal subunit where proteins L1, L3, L13, L15, L16, L18, L19, L22, and L24 significantly stimulated trans splicing. More moderate stimulation of trans splicing was observed with L4 and L17. Next we asked if a combination of two proteins further increases the splicing rates. Ribosomal proteins L4 and L24 both assemble to a short fragment within 23S rRNA (Stelzl and Nierhaus 2001) suggesting that together they might act cooperatively. However, addition of L4 together with L24 did not further stimulate trans splicing (data not shown).
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). Ribosomal protein L1 consists of two globular domains and is localized to the stalk region near the E-site. Previous studies indicate that this is a highly flexible region of the ribosome (Stark et al. 2000; Uchiumi et al. 2002). It has been further suggested that L1 plays a role in facilitating E-site tRNA release, an activity that may be facilitated by RNA chaperone activity (Agrawal et al. 1999; Gomez-Lorenzo et al. 2000; Harms et al. 2001; Yusupov et al. 2001). Finally, L1 is a translational repressor that regulates expression of its own mRNA in E. coli (Nomura et al. 1984; Nikonov et al. 1996). While general models for translation control by several ribosomal proteins are based on sequestration of the Shine–Dalgarno region, it is possible that these proteins directly resolve RNA structures to promote their own repressor activities. A similar model has recently been proposed for another RNA chaperone, Hfq, which binds to and resolves RNA structural elements (Geissmann and Touati 2004). Ribosomal protein L3 is one of the early assembling proteins (Rohl and Nierhaus 1982) and is composed of a globular domain and an unstructured extension (Ban et al. 2000; Harms et al. 2001). The unstructured extension has a methylated glutamine residue and the loss of this modification results in cold-sensitive ribosome assembly (Chang and Chang 1975; Lhoest and Colson 1981). Perhaps L3 directly facilitates RNA rearrangements in this region of the ribosome that are critical in preventing the long-term sampling of cold-sensitive metastable states. Finally, like L1, ribosomal protein L4 is a negative transcriptional and translational regulator of its own operon (Lindahl and Zengel 1979; Yates et al. 1980; Zengel et al. 1980), consisting of a globular domain with a long unstructured extension, like L3 (Ban et al. 2000; Harms et al. 2001). Both L4 and L22 are located along the exit tunnel of the ribosome, suggesting potential roles in regulating the extrusion of the growing peptide (Nissen et al. 2000; Gabashvili et al. 2001).
Most of the ribosomal proteins are located at the periphery of the ribosome and are thought to play critical roles both in assembly and stabilization of the ribosome structure. Some of the ribosomal proteins that have significant RNA chaperone activity in our assay assemble at early stages of reconstitution (e.g., L3 and L4) (Nierhaus 1991). Other proteins that we have identified are involved at later stages during assembly (e.g., L15, L16) (Franceschi and Nierhaus 1990). It is easy to imagine that RNA chaperone activity could be important at all stages of assembly, ensuring that the RNA does not become trapped in nonnative structures. It is also possible that the ribosomal proteins play critical roles in facilitating RNA rearrangements during translation, and that these movements depend on the RNA chaperone-like properties of these unusual proteins. We propose that the RNA chaperone activity of the ribosomal proteins identified here, besides preventing the formation of misfolded structures during assembly, could be more generally critical to the dynamic rearrangements of the ribosome during tRNA selection, peptide bond formation, and translocation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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). The resuspended pellet or supernatant was dialyzed at 4°C overnight into the respective FPLC buffer (Table 1). The lysate was then purified over a resource S cation column (or a resource Q anion column). The proteins were eluted over a salt gradient (20 mM KCl–1000 mM KCl) and dialyzed into storage buffer (20 mM Tris at pH 7.4, 4 mM MgAc2, 400 mM NH4Cl, 0.2 mM EDTA, 5 mM ß-mercaptoEtOH) except for L4, which was stored in buffer C (Table 1) and dialyzed into storage buffer prior to usage. The purity of the ribosomal proteins was tested on Coomassie-stained SDS urea gels. The ribosomal proteins were further evaluated for RNase activity by incubating the ribosomal protein with transcribed RNA and testing RNA degradation by separation on a 5% polyacrylamide gel (PAGE). Ribosomal proteins that still showed a significant amount of RNase activity were again purified either at the same pH or at a slightly increased pH and were eluted with the increasing salt gradient (20 mM KCl–1000 mM KCl). Then the proteins were dialyzed into the storage buffer (20 mM Tris at pH 7.4, 4 mM MgAc2, 400 mM NH4Cl, 0.2 mM EDTA, 5 mM ß-mercaptoEtOH).
In vitro transcription
The plasmids for H1 (exon 1 + 5' intron) and H2 (3' intron + exon 2) were linearized with SalI (for H1) and BamHI (for H2). Construct H1 consists of 549 nt of exon 1 and 131 nt of the 5' part of the intron (total 680 nt). Construct H2 consists of 147 nt of the 3' half of the intron and 23 nt of exon 2 (total 170 nt). The RNAs H1 and H2 were transcribed with 40 mM Tris (pH 7.0), 26 mM MgCl2, 3 mM spermidine, 5 mM ATP, 5 mM GTP, 5 mM CTP, 2.5 mM UTP, 2.5 mM 35S--UTP, 10 mM DTT, T7 RNA polymerase at 37°C for 3 h, followed by 30 min DNase digest and purification of the transcripts over a 5% polyacrylamide gel (PAGE).
Trans splicing assay
We incubated 200 nM H1 and 200 nM H2 transcripts 1 min at 95°C and cooled them to either 55°C (for the positive control) or 37°C. Next, splicing buffer (4 mM Tris at pH 7.4, 3 mM MgCl2, 0.4 mM spermidine, 4 mM DTT final concentration) and 0.33 pmol 32P-GTP were added. Additionally, either the respective ribosomal protein was added to a final concentration of 2 µM or the same quantity of ribosomal storage buffer (20 mM Tris at pH 7.4, 4 mM MgAc2, 400 mM NH4Cl, 0.2 mM EDTA, 5 mM ß-mercaptoEtOH) was added. The reactions were incubated at either 55°C (positive control) or at 37°C and aliquots were stopped by adding a final concentration of 40 mM EDTA and 300 µg/mL tRNA. The samples were phenol-CHCl3 extracted, precipitated, and loaded on 5% polyacrylamide gels. Bands corresponding to the product of the first step of splicing (intron 1 with 5' added guanosine) were measured by PhosphorImager and relative rates were calculated for each gel by setting the positive control to 1 (trans splicing at 55°C in the absence of ribosomal proteins) and subtracting the negative control (trans splicing at 37°C in the absence of ribosomal proteins) from each obtained rate on each gel by the formula: (nx - n37)/(n55 - n37), nx being the relative splicing rate in the presence of the respective ribosomal protein, n55 being the relative splicing rate at 55°C in the absence of ribosomal proteins, and n37 being the relative splicing rate at 37°C in the absence of a ribosomal protein.
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ACKNOWLEDGMENTS |
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Footnotes |
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Received July 7, 2004; accepted September 13, 2004.
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REFERENCES |
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