A Small Protein Unique to Bacteria Organizes rRNA Tertiary Structure Over an Extensive Region of the 50 S Ribosomal Subunit

A number of small, basic proteins penetrate into the structure of the large subunit of the ribosome. While these proteins presumably aid in the folding of the rRNA, the extent of their contribution to the stability or function of the ribosome is unknown. One of these small, basic proteins is L36, which is highly conserved in Bacteria, but is not present in Archaea or Eucarya. Comparison of ribosome crystal structures shows that the space occupied by L36 in a bacterial ribosome is empty in an archaeal ribosome. To ask what L36 contributes to ribosome stability and function, we have constructed an Escherichia coli strain lacking ribosomal protein L36; cell growth is slowed by 40–50% between 30 °C and 42 °C. Ribosomes from this deletion strain sediment normally and have a full complement of proteins, other than L36. Chemical protection experiments comparing rRNA from wild-type and L36-deficient ribosomes show the expected increase in reagent accessibility in the immediate vicinity of the L36 binding site, but suggest that a cooperative network of rRNA tertiary interactions has been disrupted along a path extending 60 Å deep into the ribosome. These data argue that L36 plays a significant role in organizing 23 S rRNA structure. Perhaps the Archaea and Eucarya have compensated for their lack of L36 by maintaining more stable rRNA tertiary contacts or by adopting alternative protein–RNA interactions elsewhere in the ribosome.

Introduction

Ribosomes consist of three RNA molecules and 50–80 proteins. Although the highly conserved functional regions of the ribosome consist mostly of RNA,¹ many of the ribosomal proteins have been highly conserved across all three phylogenetic domains as well.² Thus, some ribosomal proteins must have been selected to optimize ribosome function in specific ways, which might include prevention of RNA misfolding, promotion of RNA conformational changes during the translation cycle, and stabilization of the structure of the ribosome.

One example of how ribosomal proteins have been optimized for a specific function is suggested by the prevalence of small, basic proteins that penetrate into the rRNA structure of the 50 S subunit.³ The role of these proteins is presumably to aid in the folding of rRNA; however, the extent of their contribution is not known. In order to specifically address the contribution a small, basic protein has in the ribosome, the role of one ribosomal protein, L36, will be examined in this work. L36 is the smallest bacterial ribosomal protein;⁴ it has a zinc-ribbon-like fold consisting of a small β-sheet and is highly basic (Figure 1).⁵ As can be seen in the Deinococcus radiodurans 50 S crystal structure,⁶ L36 is surrounded by several highly conserved functional regions of 23 S rRNA, including helices which stem off the peptidyl transferase center (helices 89 and 91), the L10-L11 binding domain (domain II), and the four-helix junction containing the sarcin-ricin loop (helix 95). From its position crosslinking helices 89 and 91, L36 sits on the divide between two symmetry-related regions extending out from the peptidyl transferase center.⁷

L36 is a highly conserved bacterial protein found in all branches of eubacteria as well as in mitochondria and chloroplasts.⁸ Alignments of L36 sequences illustrate that basic residues (12 Arg and Lys in Escherichia coli) tend to be conserved. In addition, the CCCH zinc-binding motif is conserved in ∼75% of the 160 completed bacterial genomes in which L36 was found and in all known mitochondrial and chloroplast sequences. Although L36 is conserved in bacteria, it is not present in archaeal or eucaryal genomes.⁸ Examination of 50 S subunit crystal structures from an Archaea and a Bacteria shows that the space occupied by L36 in the bacterial structure is empty in the archaeal structure (Figure 1). These observations lead to the question of how important L36 is for ribosome function: might it make such a marginal contribution that Archaea and Eucarya have not retained an equivalent protein, or might it serve an important role which Eucarya and Archaea have achieved by alternative means.

To ask what role L36 plays in the ribosome in bacteria, an E. coli strain lacking ribosomal protein L36 was constructed. Removal of L36 from the ribosome affects ribosome function strongly and causes extensive disruptions of the rRNA tertiary structure. The disruptions extend from the surface to deep within the ribosome, suggesting that the rRNA fold is highly cooperative and relies on the strategic placement of positive charges for its maintenance. We argue from these data that L36 has a specific and important role in the organization of the 23 S rRNA, and speculate that Archaea and Eucarya have used other means to achieve the same degree of stabilization.