Evolution of Ribosomal Protein S14 Demonstrated by the Reconstruction of Chimeric Ribosomes in Bacillus subtilis

Abstract

Ribosomal protein S14 can be classified into three types. The first, the C+ type has a Zn2+ binding motif and is ancestral. The second and third are the C- short and C- long types, neither of which contain a Zn2+ binding motif and which are ca. 90 residues and 100 residues in length, respectively. In the present study, the C+ type S14 from Bacillus subtilis ribosomes (S14BsC+) were completely replaced by the heterologous C- long type of S14 from Escherichia coli (S14Ec) or Synechococcus elongatus (S14Se). Surprisingly, S14Ec and S14Se were incorporated fully into 70S ribosomes in B. subtilis. However, the growth rates as well as the sporulation efficiency of the mutants harboring heterologous S14 were significantly decreased. In these mutants, the polysome fraction was decreased and the 30S and 50S subunits accumulated unusually, indicating that cellular translational activity of these mutants was decreased. In vitro analysis showed a reduction in the translational activity of the 70S ribosome fraction purified from these mutants. The abundance of ribosomal proteins S2 and S3 in the 30S fraction in these mutants was reduced while that of S14 was not significantly decreased. It seems likely that binding of heterologous S14 changes the structure of the 30S subunit, which causes a decrease in the assembly efficiency of S2 and S3 which are located near the binding site of S14. Moreover, we found that S3 from S. elongatus cannot function in B. subtilis unless S14Se is present. IMPORTANCE S14, an essential ribosomal protein, may have evolved to adapt bacteria to zinc-limited environments by replacement of a zinc-binding motif with a zinc independent sequence. It was expected that the bacterial ribosome would be tolerant to replacement of S14, because of the previous prediction that the spread of C- type S14 involved horizontal gene transfer. In this study, we completely replaced the C+ type of S14 in B. subtilis ribosome with the heterologous C- long type of S14 and characterized the resulting chimeric ribosomes. Our results suggest that the B. subtilis ribosome is permissive for the replacement of S14 but coevolution of S3 might be required to utilize the C- long type of S14 more effectively.

Ribosomes lacking bS21 gain function to regulate protein synthesis inFlavobacterium johnsoniae

“…When cells are starved of zinc, yhzA , ytiA and rpmGC are derepressed, the C- forms of uS14, bL31 and bL33 are produced, and these proteins substitute for their C+ equivalents in the ribosome. This allows Zn 2+ to be liberated for more critical use and enables new ribosomes to be made with fewer Zn 2+ ions ( 12–15 ). Given the abundance of ribosomes (7000–70 000 per cell, depending on growth rate ( 16 , 17 )), exchangeable RPs represent a substantial reservoir of Zn 2+ for the cell.…”

Sarecycline inhibits protein translation inCutibacterium acnes70S ribosome using a two-site mechanism

“…Most bacteria have only one variant of uS14. It was proposed that the loss of the Zn 2+ -binding motif evolved to adapt bacteria to zinc-limited environments ( 49 ). Similar to M. tuberculosis , C. acnes genome has both zinc and zinc-free variants for six ribosomal proteins ( Supplementary Table S2 ).…”

‘Tuning’ of ribosome levels mediated by RNase I and hibernating ribosomes

“…Despite the gene for uS14 being essential, the function of uS14 in translation remains elusive. The uS14 protein is classified into three groups according to the presence or absence of a zinc-binding motif, and E. coli uS14 belongs to the C-long type 65 . Compared to the crystal structure of uS14 in Thermus thermophilus 70S ribosome (PDB 4V8H 34 ) , which belongs to the C+ type, E. coli uS14 has a distinctive domain with an internal insertion containing the contact site to RNase I (Extended Data Fig.…”

“…No homologous protein of RNase I has been found in T. thermophilus, suggesting that E. coli uS14 may have evolved independently, along with its interaction with RNase I. In addition, C+/-paralogue pairs for uS14 are conserved in some bacteria, and their functionality differences have been reported [65][66][67] . These results support that uS14 is a susceptible region in the evolutionary process for gaining diverse functions.…”