Purification, cloning, and properties of the 16S RNA pseudouridine 516 synthase from Escherichia coli

Abstract

Pseudouridine (psi) is commonly found in both small and large subunit ribosomal RNAs of prokaryotes and eukaryotes. In Escherichia coli small subunit RNA, there is only one psi, at position 516, in a region of the RNA known to be involved in codon recognition [Bakin et al. (1994) Nucleic Acids Res. 22, 3681-3684]. To assess the function of this single psi residue, the enzyme catalyzing its formation was purified and cloned. The enzyme contains 231 amino acids and has a calculated molecular mass of 25,836 Da. It converts U516 in E. coli 16S RNA transcripts into psi but does not modify any other position in this RNA. It does not react with free unmodified 16S RNA at all, and only poorly with 30S particles containing unmodified RNA. The preferred substrate is an RNA fragment from residues 1 to 678 which has been complexed with 30S ribosomal proteins. The yield varied from 0.6 to 1.0 mol of psi/mol of RNA, depending on the preparation. Free RNA(1-678) was inactive, as was RNA(1-526) and the RNP particle made from it. 23S RNA and tRNAVal transcripts were also inactive. These results suggest that psi formation in vivo occurs at an intermediate stage of 30S assembly. The gene is located at 47.1 min immediately 5' to, and oriented in the same direction as, the bicyclomycin resistance gene. The gene was cloned behind a (His)6 leader for affinity purification. Virtually all of the overexpressed protein was found in inclusion bodies but could be purified to homogeneity on a Ni2+(-) containing resin. Over 200 mg of pure protein could be obtained from a liter of cell culture. Amino acid sequence comparison revealed the existence of a gene in Bacillus subtilis with a similar sequence, and psi sequence analysis established that B. subtilis has the equivalent of psi 516 in its small subunit rRNA. On the other hand, no common sequence motifs could be detected among this enzyme and the two tRNA psi synthases which have been cloned up to now.

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

“…So far, we have identified three such genes, rsuA (15), rsmB, 2 and in this work, rsmC. rsmA (ksgA) was already known (8).…”

“…Perhaps the ability of the m 5 C967 methyltransferase to react with free RNA, but not with 30 S subunits, is the exception to the rule, and most or all of the other 16 S RNA-modifying enzymes require either a ribonucleoprotein or a complete 30 S subunit. RsuA, which makes the single pseudouridine in 16 S RNA, also has a specific requirement for a particular RNP particle (15).…”

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

“…Although the structure of pseudouridine (⌿) was determined almost 40 years ago (Cohn, 1960), its role in RNA remains an enigma+ Despite the fact that ⌿ is found in all classes of RNA that must maintain a tertiary structure for proper function, namely tRNA (Sprinzl et al+, 1998), rRNA (Maden, 1990), and sn(o)RNA (Gu et al+, 1998;Massenet et al+, 1998), the function of ⌿ in these molecules has remained elusive+ The presence of ⌿ in rRNA, especially in the large subunit (LSU) RNA, is noteworthy+ There, the ⌿ residues cluster in or near the peptidyl transferase center of all organisms studied, despite large variations in the total number of ⌿ found in the RNA (Ofengand & Bakin, 1997)+ ⌿ is formed at the polynucleotide level by isomerization of selected uridines in an enzyme-catalyzed reaction requiring neither added cofactors nor an energy source (reviewed in Ofengand & Fournier, 1998)+ Locating and disrupting the genes coding for ⌿ synthases should, therefore, result in the absence of specific ⌿ residues, assuming that individual or subsets of ⌿ are formed by distinct synthases+ This is true for the cloned synthases for tRNA (Kammen et al+, 1988;Nurse et al+, 1995;Becker et al+, 1997;Grosjean et al+, 1997;Lecointe et al+, 1998), and appears to be the case for rRNA as well (Wrzesinski et al+, 1995a,b)+ In Escherichia coli, there are 9 ⌿ in the LSU RNA (Bakin & Ofengand, 1993;Bakin et al+, 1994b), and one in the small subunit (SSU) RNA (Bakin et al+, 1994a)+ To understand the function of ⌿ in rRNA, we have embarked on a program to block the formation of specific ⌿ residues by interfering with production of the enzymes required for their biosynthesis+ We previously identified a synthase, RsuA, specific for the single ⌿ in SSU RNA (Wrzesinski et al+, 1995a) and another, RluA, specific for ⌿746 in LSU RNA (Wrzesinski et al+, 1995b)+ We recently identified an additional synthase, RluC, which forms ⌿955, ⌿2504, and ⌿2580 (Conrad et al+, 1998; see Table 1)+ Here, we describe another synthase, RluD, the product of the sfhB (previously known as yfiI ) gene, renamed rluD, which is solely responsible in vivo for synthesis of ⌿1911, ⌿1915, and ⌿1917+ Two of these, ⌿1915 and ⌿1917, are found in the equivalent location in the LSU RNA of all organisms examined, which include representatives from the Prokarya, Eukarya, Archaea, mitochondria, and chloroplasts (Ofengand & Bakin, 1997)+ Disruption of the rluD gene and consequent lack of these three...…”

Transglycosylation: A mechanism for RNA modification (and editing?)

“…To date, five families of pseudouridine synthases have been isolated [68][69][70][71][72][73]. Two families of enzymes appear to catalyze formation of pseudouridine in tRNA, while the others appear to be responsible for pseudouridine formation in rRNA.…”