Enhanced expression of Bacillus subtilis yaaA can restore both the growth and the sporulation defects caused by mutation of rplB, encoding ribosomal protein L2

A temperature-sensitive mutation in rplB, designated rplB142, encodes a missense mutation at position 142 [His (CAT) to Leu (CTT)] of Bacillus subtilis ribosomal protein L2. The strain carrying the mutation grew more slowly than the wild-type, even at low temperatures, probably due to the formation of defective 70S ribosomes and the accumulation of incomplete 50S subunits (50S* subunits). Gel analysis indicated that amounts of L2 protein and also of L16 protein were reduced in ribosomes prepared from the rplB142 mutant 90 min after increasing the growth temperature to 45 °C. These results suggest that the assembly of the L16 protein into the 50S subunit requires the native L2 protein. The H142L mutation in the defective L2 protein affected sporulation as well as growth, even at the permissive temperature. A suppressor mutation that restored both growth and sporulation of the rplB142 mutant at low temperature was identified as a single base deletion located immediately upstream of the yaaA gene that resulted in an increase in its transcription. Furthermore, genetic analysis showed that enhanced synthesis of YaaA restores the functionality of L2 (H142L) by facilitating its assembly into 50S subunits.

The Ribosomal Protein L2 Interacts with the RNA Polymerase α Subunit and Acts as a Transcription Modulator in Escherichia coli

ABSTRACT Identification of interacting proteins in stable complexes is essential to understand the mechanisms that regulate cellular processes at the molecular level. Transcription initiation in prokaryotes requires coordinated protein-protein and protein-DNA interactions that often involve one or more transcription factors in addition to RNA polymerase (RNAP) subunits. The RNAP α subunit (RNAPα) is a key regulatory element in gene transcription and functions through direct interaction with other proteins to control all stages of this process. A clear description of the RNAPα protein partners should greatly increase our understanding of transcription modulation. A functional proteomics approach was employed to investigate protein components that specifically interact with RNAPα. A tagged form of Escherichia coli RNAPα was used as bait to determine the molecular partners of this subunit in a whole-cell extract. Among other interacting proteins, 50S ribosomal protein L2 (RPL2) was clearly identified by mass spectrometry. The direct interaction between RNAPα and RPL2 was confirmed both in vivo and in vitro by performing coimmunoprecipitation and bacterial two-hybrid experiments. The functional role of this interaction was also investigated in the presence of a ribosomal promoter by using a β-galactosidase gene reporter assay. The results clearly demonstrated that RPL2 was able to increase β-galactosidase expression only in the presence of a specific ribosomal promoter, whereas it was inactive when it was assayed with an unrelated promoter. Interestingly, other ribosomal proteins (L1, L3, L20, and L27) did not have any effect on rRNA expression. The findings reported here strongly suggest that in addition to its role in ribosome assembly the highly conserved RPL2 protein plays a specific and direct role in regulation of transcription.