James Ofengand

Ribosomal RNA pseudouridines and pseudouridine synthases

Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui pseudouridines on the three‐dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA–protein complex. No unique specific role for ribosomal pseudouridines has been identified. We propose that pseudouridines function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.

A pseudouridine synthase required for the formation of two universally conserved pseudouridines in ribosomal RNA is essential for normal growth of Escherichia coli

Escherichia coli rRNA contains 10 pseudouridines of unknown function. They are made by synthases, each of which is specific for one or more pseudouridines. Here we show that the sfhB (yfil) ORF of E. coli is a pseudouridine synthase gene by cloning, protein overexpression, and reaction in vitro with rRNA transcripts. Gene disruption by miniTn10(cam) insertion revealed that this synthase gene, here renamed rluD, codes for a synthase which is solely responsible in vivo for synthesis of the three pseudouridines clustered in a stem-loop at positions 1911, 1915, and 1917 of 23S RNA. The absence of RluD results in severe growth inhibition. Both the absence of pseudouridine and the growth defect could be reversed by insertion of a plasmid carrying the rluD gene into the mutant cell, clearly linking both effects to the absence of RIuD. This is the first report of a major physiological defect due to the deletion of any pseudouridine synthase. Growth inhibition may be due to the lack of one or more of the 23S RNA pseudouridines made by this synthase since pseudouridines 1915 and 1917 are universally conserved and are located in proximity to the decoding center of the ribosome where they could be involved in modulating codon recognition.

Deletion of the Escherichia coli pseudouridine synthase gene truB blocks formation of pseudouridine 55 in tRNA in vivo, does not affect exponential growth, but confers a strong selective disadvantage in competition with wild-type cells

Previous work from this laboratory (Nurse et al., RNA, 1995, 1:102-112) established that TruB, a pseudouridine (psi) synthase from Escherichia coli, was able to make psi55 in tRNA transcripts but not in transcripts of full-length or fragmented 16S or 23S ribosomal RNAs. By deletion of the truB gene, we now show that TruB is the only protein in E. coli able to make psi55 in vivo. Lack of TruB and psi55 did not affect the exponential growth rate but did confer a strong selective disadvantage on the mutant when it was competed against wild-type. The negative selection did not appear to be acting at either the exponential or stationary phase. Transformation with a plasmid vector conferring carbenicillin resistance and growth in carbenicillin markedly increased the selective disadvantage, as did growth at 42 degrees C, and both together were approximately additive such that three cycles of competitive growth sufficed to reduce the mutant strain to approximately 0.2% of its original value. The most striking finding was that all growth effects could be reversed by transformation with a plasmid carrying a truB gene coding for a D48C mutation in TruB. Direct analysis showed that this mutant did not make psi55 under the conditions of the competition experiment. Therefore, the growth defect due to the lack of TruB must be due to the lack of some other function of the protein, possibly an RNA chaperone activity, but not to the absence of psi55.

Functional Effect of Deletion and Mutation of the Escherichia coli Ribosomal RNA and tRNA Pseudouridine Synthase RluA

The Escherichia coli generluA, coding for the pseudouridine synthase RluA that forms 23 S rRNA pseudouridine 746 and tRNA pseudouridine 32, was deleted in strains MG1655 and BL21/DE3. The rluA deletion mutant failed to form either 23 S RNA pseudouridine 746 or tRNA pseudouridine 32. Replacement of rluA in trans on a rescue plasmid restored both pseudouridines. Therefore, RluA is the sole protein responsible for the in vivo formation of 23 S RNA pseudouridine 746 and tRNA pseudouridine 32. Plasmid rescue of bothrluA − strains using an rluA gene carrying asparagine or threonine replacements for the highly conserved aspartate 64 demonstrated that neither mutant could form 23 S RNA pseudouridine 746 or tRNA pseudouridine 32 in vivo, showing that this conserved aspartate is essential for enzyme-catalyzed formation of both pseudouridines. In vitro assays using overexpressed wild-type and mutant synthases confirmed that only the wild-type protein was active despite the overexpression of wild-type and mutant synthases in approximately equal amounts. There was no difference in exponential growth rate between wild-type and MG1655(rluA −) either in rich or minimal medium at 24, 37, or 42 °C, but when both strains were grown together, a strong selection against the deletion strain was observed.

The rluC gene of Escherichia coli codes for a pseudouridine synthase that is solely responsible for synthesis of pseudouridine at positions 955, 2504, and 2580 in 23 S ribosomal RNA.

Escherichia coli ribosomal RNA contains 10 pseudouridines, one in the 16 S RNA and nine in the 23 S RNA. Previously, the gene for the synthase responsible for the 16 S RNA pseudouridine was identified and cloned, as was a gene for a synthase that makes a single pseudouridine in 23 S RNA. The yceCopen reading frame of E. coli is one of a set of genes homologous to these previously identified ribosomal RNA pseudouridine synthases. In this work, the gene was cloned, overexpressed, and shown to code for a pseudouridine synthase able to react with in vitro transcripts of 23 S ribosomal RNA. Deletion of the gene and analysis of the 23 S RNA from the deletion strain for the presence of pseudouridine at its nine known sites revealed that this synthase is solely responsible in vivo for the synthesis of three of the nine pseudouridine residues, at positions 955, 2504, and 2580. Therefore, this gene has been renamed rluC. Despite the absence of one-third of the normal complement of pseudouridines, there was no change in the exponential growth rate in either LB or M-9 medium at temperatures ranging from 24 to 42 °C. From this work and our previous studies, we have now identified three synthases that account for 50% of the pseudouridines in the E. coli ribosome.

A second function for pseudouridine synthases: A point mutant of RluD unable to form pseudouridines 1911, 1915, and 1917 in Escherichia coli 23S ribosomal RNA restores normal growth to an RluD-minus strain.

This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli pseudouridine synthase responsible for synthesis of 23S rRNA pseudouridines 1911, 1915, and 1917, blocks pseudouridine formation and inhibits growth. We now show that RluD mutants at the essential aspartate 139 allow these two functions of RluD to be separated. In vitro, RluD with aspartate 139 replaced by threonine or asparagine is completely inactive. In vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with aspartate 139 replaced by threonine or asparagine. Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrated the lack of these pseudouridines. Pseudoreversion, which has previously been shown to restore growth without pseudouridine formation by mutation at a distant position on the chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.

Purification, Cloning, and Characterization of the 16 S RNA m2G1207 Methyltransferase from Escherichia coli

The methyltransferase that forms m2G1207 in Escherichia coli small subunit rRNA has been purified, cloned, and characterized. The gene was identified from the N-terminal sequence of the purified enzyme as the open reading frame yjjT (SWISS-PROT accession numberP39406). The gene, here renamed rsmC in view of its newly established function, codes for a 343-amino acid protein that has homologs in prokaryotes, Archaea, and possibly also in lower eukaryotes. The enzyme reacted well with 30 S subunits reconstituted from 16 S RNA transcripts and 30 S proteins but was almost inactive with the corresponding free RNA. By hybridization and protection of appropriate segments of 16 S RNA that had been extracted from 30 S subunits methylated by the enzyme, it was shown that of the three naturally occurring m2G residues, only m2G1207 was formed. Whereas close to unit stoichiometry of methylation could be achieved at 0.9 mm Mg2+, both 2 mmEDTA and 6 mm Mg2+ markedly reduced the level of methylation, suggesting that the optimal substrate may be a ribonucleoprotein particle less structured than a 30 S ribosome but more so than free RNA.

Crystallization and characterization of a fragment of pseudouridine synthase RluC from Escherichia coli

RluC from E. coli is the enzyme responsible for catalyzing the isomerization of uridines 955, 2504 and 2580 in 23S rRNA to pseudouridine. Histidine-tagged RluC was cloned, overexpressed and purified by nickel-affinity chromatography. A proteolytically derived fragment of the enzyme consisting of residues 89-319 has been shown to retain catalytic activity. Crystals of this fragment, grown by precipitation with sodium acetate at pH 8.0, belong to space group P321, with unit-cell dimensions a = b = 97.1, c = 86.3 A and have two molecules in the crystallographic asymmetric unit. The flash-frozen crystals diffract X-rays to at least 2.3 A resolution and appear suitable for crystal structure determination.

PURIFICATION AND CRYSTALLIZATION OF ESCHERICHIA COLI PSEUDOURIDINE SYNTHASE RLUD

RluD is the pseudouridine (Psi) synthase responsible for forming Psi1911, Psi1915 and Psi1917 in Escherichia coli 23S RNA. Out of the 11 Psi synthases in E. coli, only cells lacking RluD show a severe growth defect. In addition, RluD belongs to the RluA family of Psi synthases, one of the two remaining families without a representative crystal structure. In this paper, the crystallization of selenomethionine-substituted RluD by the hanging-drop method is reported. The crystals diffract to 1.9 A and belong to space group P4(3)2(1)2, with unit-cell parameters a = b = 75.14, c = 181.81 A. Synchrotron radiation was used on a single crystal to collect a complete multiwavelength anomalous dispersion (MAD) data set to 2.0 A resolution.