The FtsJ/RrmJ heat shock protein of Escherichia coli is a 23 S ribosomal RNA methyltransferase

Ribosomal RNAs undergo several nucleotide modifications including methylation. We identify FtsJ, the first encoded protein of the ftsJ-hflB heat shock operon, as an Escherichia coli methyltransferase of the 23 S rRNA. The methylation reaction requires S-adenosylmethionine as donor of methyl groups, purified FtsJ or a S(150) supernatant from an FtsJ-producing strain, and ribosomes from an FtsJ-deficient strain. In vitro, FtsJ does not efficiently methylate ribosomes purified from a strain producing FtsJ, suggesting that these ribosomes are already methylated in vivo by FtsJ. FtsJ is active on ribosomes and on the 50 S ribosomal subunit, but is inactive on free rRNA, suggesting that its natural substrate is ribosomes or a pre-ribosomal ribonucleoprotein particle. We identified the methylated nucleotide as 2'-O-methyluridine 2552, by reverse phase high performance liquid chromatography analysis, boronate affinity chromatography, and hybridization-protection experiments. In view of its newly established function, FtsJ is renamed RrmJ and its encoding gene, rrmJ.     

Active site in RrmJ,a heat shock-induced methyltransferase

The heat shock protein RrmJ (FtsJ), highly conserved from eubacteria to eukarya, is responsible for the 2'-O-ribose methylation of the universally conserved base U2552 in the A-loop of the 23 S rRNA. Absence of this methylation, which occurs late in the maturation process of the ribosome, appears to cause the destabilization and premature dissociation of the 50 S ribosomal subunit. To understand the mechanism of 2'-O-ribose methyltransfer reactions, we characterized the enzymatic parameters of RrmJ and conducted site-specific mutagenesis of RrmJ. A structure based sequence alignment with VP39, a structurally related 2'-O-methyltransferase from vaccinia virus, guided our mutagenesis studies. We analyzed the function of our RrmJ mutants in vivo and characterized the methyltransfer reaction of the purified proteins in vitro. The active site of RrmJ appears to be formed by a catalytic triad consisting of two lysine residues, Lys-38 and Lys-164, and the negatively charged residue Asp-124. Another highly conserved residue, Glu-199, that is present in the active site of RrmJ and VP39 appears to play only a minor role in the methyltransfer reaction in vivo. Based on these results, a reaction mechanism for the methyltransfer activity of RrmJ is proposed.     

Structure of Hsp15 reveals a novel RNA-binding motif

We have solved the crystal structure of the heat shock protein Hsp15, a newly isolated and very highly inducible heat shock protein that binds the ribosome. Comparison of its structure with those of two RNA-binding proteins, ribosomal protein S4 and threonyl-tRNA synthetase, reveals a novel RNA-binding motif. This newly recognized motif is remarkably common, present in at least eight different protein families that bind RNA. The motif's surface is populated by conserved, charged residues that define a likely RNA-binding site. An intriguing pattern emerges: stress proteins, ribosomal proteins and tRNA synthetases repeatedly share a conserved motif. This may imply a hitherto unrecognized functional similarity between these three protein classes.     

Overexpression of two different GTPases rescues a null mutation in a heat-induced rRNA methyltransferase

The Escherichia coli RrmJ (FtsJ) heat shock protein functions as an rRNA methyltransferase that modifies position U2552 of 23S rRNA in intact 50S ribosomal subunits. An in-frame deletion of the rrmJ (ftsJ) gene leads to severe growth disadvantages under all temperatures tested and causes significant accumulation of ribosomal subunits at the expense of functional 70S ribosomes. To investigate whether overexpression of other E. coli genes can restore the severe growth defect observed in rrmJ null mutants, we constructed an overexpression library from the rrmJ deletion strain and cloned and identified the E. coli genes that were capable of rescuing the rrmJ mutant phenotype. Our intention was to identify other methylases whose specificities overlapped enough with that of RrmJ to allow complementation when overexpressed. To our great surprise, no methylases were found by this method; rather, two small GTPases, Obg (YhbZ) and EngA, when overexpressed in the rrmJ deletion strains, were found to restore the otherwise severely impaired ribosome assembly process and/or stability of 70S ribosomes. 50S ribosomal subunits prepared from these overexpressing strains were shown to still serve as in vitro substrates for purified RrmJ, indicating that the 23S rRNA likely was still lacking the highly conserved Um2552 modification. The apparent lack of this modification, however, no longer caused ribosome defects or a growth disadvantage. Massive overexpression of another related small GTPase, Era, failed to rescue the growth defects of an rrmJ strain. These findings suggest a hitherto unexpected connection between rRNA methylation and GTPase function, specifically that of the two small GTPases Obg and EngA.     

Solution structure of GSP13 from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> exhibits an S1 domain related to cold shock proteins

GSP13 encoded by gene yugI is a σ^B-dependent general stress protein in Bacillus subtilis, which can be induced by heat shock, salt stress, ethanol stress, glucose starvation, oxidative stress and cold shock. Here we report the solution structure of GSP13 and it is the first structure of S1 domain containing protein in Bacillus subtilis. The structure of GSP13 mainly consists of a typical S1 domain along with a C-terminal 50-residue flexible tail, different from the other known S1 domain containing proteins. Comparison with other S1 domain structures reveals that GSP13 has a conserved RNA binding surface, and it may function similarly to cold shock proteins in response to cold stress.     

Loss of 4.5S RNA induces the heat shock response and lambda prophage in Escherichia coli.

During depletion of 4.5S RNA, cells of Escherichia coli displayed a heat shock response that was simultaneous with the first detectable effect on ribosome function and before major effects on cell growth. Either 4.5S RNA is involved directly in regulating the heat shock response, or this particular impairment of protein synthesis uniquely induces the heat shock response. Several hours later, lambda prophage was induced and the cells lysed.     

Hsp15: a ribosome-associated heat shock protein

We are analyzing highly conserved heat shock genes of unknown or unclear function with the aim of determining their cellular role. Hsp15 has previously been shown to be an abundant nucleic acid-binding protein whose synthesis is induced massively at the RNA level upon temperature upshift. We have now identified that the in vivo target of Hsp15 action is the free 50S ribosomal subunit. Hsp15 binds with very high affinity (K(D) <5 nM) to this subunit, but only when 50S is free, not when it is part of the 70S ribosome. In addition, the binding of Hsp15 appears to correlate with a specific state of the mature, free 50S subunit, which contains bound nascent chain. This provides the first evidence for a so far unrecognized abortive event in translation. Hsp15 is suggested to be involved in the recycling of free 50S subunits that still carry a nascent chain. This gives Hsp15 a very different functional role from all other heat shock proteins and points to a new aspect of translation.     

Putative 65 kDa protein of beet yellows closterovirus is a homologue of HSP70 heat shock proteins

A portion of the RNA genome of beet yellows closterovirus (BYV) has been sequenced encompassing a complete long open reading frame (ORF) potentially encoding a 65 kDa protein. The sequence of this putative protein was strikingly similar to those of HSP70-related heat shock proteins. The counterparts of all the eight segments strongly conserved in HSP70s could be confidently identified in the BYV 65 kDa protein. It is suggested that some of these segments might be the ATP-binding site(s) and that, similarly to the heat shock proteins, the 65 kDa is probably ATP-binding. Generally, however, the divergence between the 65 kDa sequence and the sequences of the HSP70s was much more pronounced than that between any two members of the latter family, allowing a clearer delineation of clusters of conserved residues that might be crucial for protein function. It is suggested that these observations will be helpful in functional dissection of the proteins of the HSP70 family. Analysis of the sequence of a portion of the ORF found upstream from the 65 kDa ORF showed that the C-terminal domain of the encoded protein could be an RNA-dependent RNA polymerase closely related to those of tricornaviruses, a family of RNA plant viruses with three component genomes.     

Is 20S RNA naked?

The 20S RNA of Saccharomyces cerevisiae is a single-stranded, circular RNA virus. A previous study suggested that this RNA is part of a 32S ribonucleoprotein particle, being associated with multiple copies of a 23-kilodalton protein. We show here that this protein is, in fact, the chromosome-encoded heat shock protein Hsp26. Furthermore, it is apparently not associated with 20S RNA and plays no obvious role in the life cycle of the virus.     

Substrate binding analysis of the 23S rRNA methyltransferase RrmJ

The 23S rRNA methyltransferase RrmJ (FtsJ) is responsible for the 2'-O methylation of the universally conserved U2552 in the A loop of 23S rRNA. This 23S rRNA modification appears to be critical for ribosome stability, because the absence of functional RrmJ causes the cellular accumulation of the individual ribosomal subunits at the expense of the functional 70S ribosomes. To gain insight into the mechanism of substrate recognition for RrmJ, we performed extensive site-directed mutagenesis of the residues conserved in RrmJ and characterized the mutant proteins both in vivo and in vitro. We identified a positively charged, highly conserved ridge in RrmJ that appears to play a significant role in 23S rRNA binding and methylation. We provide a structural model of how the A loop of the 23S rRNA binds to RrmJ. Based on these modeling studies and the structure of the 50S ribosome, we propose a two-step model where the A loop undocks from the tightly packed 50S ribosomal subunit, allowing RrmJ to gain access to the substrate nucleotide U2552, and where U2552 undergoes base flipping, allowing the enzyme to methylate the 2'-O position of the ribose.     

Requirement of messenger RNA synthesis for the first division in heat synchronized Tetrahymena

When Tetrahymena are starved during the heat synchronization treatment, they synthesize a small amount of transfer RNA and DNA-like RNA containing poly A, but no ribosomal RNA and still retain the capacity to divide synchronously. Analysis with MAK chromatography revealed that the DNA-like RNA is eluted almost entirely as tenaciously bound, DNA-like RNA. SDS-sucrose gradient centrifugation revealed that the DNA-like RNA is heterogeneous in size with a dominant peak sedimenting at about 17S. The peak fraction containing poly A sediments at about 15S.A good correlation has been established between the percentage of cell division and the synthesis of either tenaciously bound, DNA-like RNA or RNase-resistant RNA using various concentrations of actinomycin D. Actinomycin D treatment causes little delay in the initiation of furrowing in division but prolongs the furrowing process. In the present system, the critical addition time with actinomycin D (50 μg/ml) is about 30 min after the end of the last heat shock (EH).The present data suggest that the synthesis of messenger-like RNA containing poly A is required after the last heat shock (approx. 30–40 min after EH) for the first division in heat synchronized Tetrahymena. This RNA synthesis appears to be related to the furrowing process.