Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK: insights into tRNA recognition.

1-Methyladenosine (m1A) is a modified nucleoside found at positions 9, 14, 22 and 58 of tRNAs, which arises from the transfer of a methyl group onto the N1-atom of adenosine. The yqfN gene of Bacillus subtilis encodes the methyltransferase TrmK (BsTrmK) responsible for the formation of m1A22 in tRNA. Here, we show that BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of B. subtilis bearing an A22. In addition to a non-Watson-Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. We solved the crystal structure of BsTrmK showing an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. We used NMR chemical shift mapping to drive the docking of BstRNASer to BsTrmK in complex with its methyl-donor cofactor S-adenosyl-L-methionine (SAM). In this model, validated by methyltransferase activity assays on BsTrmK mutants, both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson-Crick R13-A22 base-pair positioning the A22 N1-atom close to the SAM methyl group.

Nus transcription elongation factors and RNase III modulate small ribosome subunit biogenesis in Escherichia coli.

Escherichia coli NusA and NusB proteins bind specific sites, such as those in the leader and spacer sequences that flank the 16S region of the ribosomal RNA transcript, forming a complex with RNA polymerase that suppresses Rho-dependent transcription termination. Although antitermination has long been the accepted role for Nus factors in rRNA synthesis, we propose that another major role for the Nus-modified transcription complex in rrn operons is as an RNA chaperone insuring co-ordination of 16S rRNA folding and RNase III processing that results in production of proper 30S ribosome subunits. This contrarian proposal is based on our studies of nusA and nusB cold-sensitive mutations that have altered translation and at low temperature accumulate 30S subunit precursors. Both phenotypes are suppressed by deletion of RNase III. We argue that these results are consistent with the idea that the nus mutations cause altered rRNA folding that leads to abnormal 30S subunits and slow translation. According to this idea, functional Nus proteins stabilize an RNA loop between their binding sites in the 5' RNA leader and on the transcribing RNA polymerase, providing a topological constraint on the RNA that aids normal rRNA folding and processing.

Ribosome biogenesis is temperature-dependent and delayed in Escherichia coli lacking the chaperones DnaK or DnaJ.

In Escherichia coli strains carrying null mutations in either the dnaK or dnaJ genes, the late stages of 30S and 50S ribosomal subunit biogenesis are slowed down in a temperature-dependent manner. At high temperature (44 degrees C), 32S and 45S particles (precursors to 50S subunits) and 21S particles (precursors to 30S subunits) accumulate. The latter are shown by 3'5' rapid amplification of cDNA ends analysis to contain unprocessed or partially processed 16S ribosomal RNA at the 5' end, but the 3' end was never processed. This implies that maturation of 16S ribosomal RNA starts at the 5'-terminus, and that the 3'-terminus is only trimmed at a later step. At normal temperatures (30 degrees C-37 degrees C), ribosome assembly in both mutants is not arrested but is significantly delayed, as shown by pulse-chase analysis. Assembly defects are partially compensated by an overexpression of other heat-shock proteins, which occurs in the absence of their negative regulator DnaK, or by a plasmid-driven overexpression of GroES/GroEL, suggesting the involvement of a network of chaperones in ribosome biogenesis.

Inhibition of chaperone-dependent bacterial ribosome biogenesis.

In Escherichia coli, the molecular chaperone HSP70 (DnaK) is necessary for 30S and 50S ribosomal subunit assembly at temperatures above 37 degrees C. Inhibitors of DnaK should therefore hinder ribosome biogenesis, in addition to all of the other DnaK-dependent cellular functions. An easily testable phenotype of DnaK is described here based on alpha-complementation of beta-galactosidase. This protein fragment complementation requires a functional DnaK in vivo, offering a suitable method for screening for DnaK inhibitors. Subsequently, it will be of great importance to check whether inhibitors of bacterial DnaK selected in this way have an effect (inhibitory or stimulatory) on the activities of eukaryotic HSP70 and HSC70 chaperones, because of the universal conservation in all biota of these chaperones in both their structural and functional properties. This question is important due to their implication in many pathways in immunology, cancer biology, and neurodegenerative disorders.