Pseudouridines and Pseudouridine Synthases of the Ribosome

Abstract

psi are ubiquitous in ribosomal RNA. Eubacteria, Archaea, and eukaryotes all contain psi, although their number varies widely, with eukaryotes having the most. The small ribosomal subunit can apparently do without psi in some organisms, even though others have as many as 40 or more. Large subunits appear to need at least one psi but can have up to 50-60. psi is made by a set of site-specific enzymes in eubacteria, and in eukaryotes by a single enzyme complexed with auxiliary proteins and specificity-conferring guide RNAs. The mechanism is not known in Archaea, but based on an analysis of the kinds of psi synthases found in sequenced archaeal genomes, it is likely to involve use of guide RNAs. All psi synthases can be classified into one of four related groups, virtually all of which have a conserved aspartate residue in a conserved sequence motif. The aspartate is essential for psi formation in all twelve synthases examined so far. When the need for psi in E. coli was examined, the only synthase whose absence caused a major decrease in growth rate under normal conditions was RluD, the synthase that makes psi 1911, psi 1915, and psi 1917 in the helix 69 end-loop. This growth defect was the result of a major failure in assembly of the large ribosomal subunit. The defect could be prevented by supplying the rluD structural gene in trans, and also by providing a point mutant gene that made a synthase unable to make psi. Therefore, the RluD synthase protein appears to be directly involved in 50S subunit assembly, possibly as an RNA chaperone, and this activity is independent of its ability to form psi. This result is not without precedent. Depletion of PET56, a 2'-O-methyltransferase specific for G2251 (E. coli numbering) in yeast mitochondria virtually blocks 50S subunit assembly and mitochondrial function (Sirum-Connolly et al. 1995), but the methylation activity of the enzyme is not required (T. Mason, pers. comm.). The absence of FtsJ, a heat shock protein that makes Um2552 in E. coli, makes the 50S subunit less stable at 1 mM Mg++ (Bügl et al. 2000) and inhibits subunit joining (Caldas et al. 2000), but, in this case, it is not yet known whether the effects are due to the lack of 2'-O-methylation or to the absence of the enzyme itself. Is there any role for the psi residues themselves? First, as noted above, the 3 psi made by RluD which cluster in the end-loop of helix 69 are highly conserved, with one being universal (Fig. 2B). In the 70S-tRNA structure (Yusupov et al. 2001), the loop of this helix containing the psi supports the anticodon arm of A-site tRNA near its juncture with the amino acid arm. The middle of helix 69 does the same thing for P-site tRNA. Unfortunately, the resolution is not yet sufficient to provide a more precise alignment of the psi residues with the other structural elements of the tRNA-ribosome complex so that one cannot yet determine what role, if any, is played by the N-1 H that distinguishes psi from U. Second, and more generally, some psi residues in the LSU appear to be near the sit...

Ribosomal RNA pseudouridines and pseudouridine synthases

“…The clustering of the 10 E. coli 8 mapped on the H. marismortui LSU is obvious, as are the more di¡use locations of the 19 H. marismortui 8. In this case also, the H. marismortui 8 locations resemble the distribution found for other eukaryotes [22]. Whereas the location of the E. coli 8 suggests a role in A site stabilization and/or function, the scatter of those in H. marismortui suggests a more general role in structural stabilization rather than one speci¢c to ligand binding.…”

“…In small subunit (SSU) RNA, there is only a single 8 in Escherichia coli and Bacillus subtilis, whereas they are numerous in the eukaryotes Saccharomyces cerevisiae, Mus musculus, and Homo sapiens. However, mapping does not show any clustering around the known functional centers of the SSU, and when placed in the three-dimensional structure of the Thermus thermophilus 30S ribosome, they appear to be distributed throughout the particle [22]. The role of 8 in the SSU is, therefore, unclear.…”

“…Table 2 also shows that an essential aspartic acid exists in all of the 8 synthases. This aspartate is found in a related common sequence context in all four E. coli families [22]. As noted in Section 1, Santi and colleagues proposed that this aspartate is involved in the catalytic mechanism [4].…”

“…All of the 8 shown in Fig. 1 have been so mapped [22]. Here we show only the results for H. marismortui and E. coli on the H. marismortui structure (Fig.…”

Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications

“…As mentioned earlier, these interactions also involve modified nucleotides of the 23S rRNA, which are likely to make these interactions stronger (ref. 39 and Jim Ofengand, personal communication). Furthermore, helix 69 of the 23S rRNA is the only component of the 50S subunit that makes contact with RRF and interacts with the decoding site within helix 44 of the 16S rRNA, the site that harbors a part of the ribosome-bound mRNA (40) and moves by Ϸ7 Å upon RRF binding (Fig.…”