Functional Sites of Ribosome Modulation Factor (RMF) Involved in the Formation of 100S Ribosome.

Hideji Yoshida,Chieko Wada,Masami Ueta,Yasushi Maki,Hideki Nakayama,Akira Wada

doi:10.3389/fmolb.2021.661691

Abstract

One of the important cellular events in all organisms is protein synthesis, which is catalyzed by ribosomes. The ribosomal activity is dependent on the environmental situation of the cell. Bacteria form 100S ribosomes, lacking translational activity, to survive under stress conditions such as nutrient starvation. The 100S ribosome is a dimer of two 70S ribosomes bridged through the 30S subunits. In some pathogens of gammaproteobacteria, such as Escherichia coli, Yersinia pestis, and Vibrio cholerae, the key factor for ribosomal dimerization is the small protein, ribosome modulation factor (RMF). When ribosomal dimerization by RMF is impaired, long-term bacterial survival is abolished. This shows that the interconversion system between active 70S ribosomes and inactive 100S ribosomes is an important survival strategy for bacteria. According to the results of several structural analyses, RMF does not directly connect two ribosomes, but binds to them and changes the conformation of their 30S subunits, thus promoting ribosomal dimerization. In this study, conserved RMF amino acids among 50 bacteria were selectively altered by mutagenesis to identify the residues involved in ribosome binding and dimerization. The activities of mutant RMF for ribosome binding and ribosome dimerization were measured using the sucrose density gradient centrifugation (SDGC) and western blotting methods. As a result, some essential amino acids of RMF for the ribosomal binding and dimerization were elucidated. Since the induction of RMF expression inhibits bacterial growth, the data on this protein could serve as information for the development of antibiotic or bacteriostatic agents.

Highlights

In the exponential growth phase of bacteria, several proteins are synthesized by a massive amount of active ribosomes through four stages: initiation, elongation, termination, and recycling (Korostelev et al, 2008)
In Escherichia coli, the expression of ribosome modulation factor (RMF) is positively regulated by the stringent response alarmone (p)ppGpp (Izutsu et al, 2001), the carbon source-sensing cAMP–cAMP receptor protein complex (Shimada et al, 2013), and the transcription factors stimulating biofilm formation such as McbR, RcdA, and SdiA (Yoshida et al, 2018)
In order to identify the functional sites in RMF involved in ribosome binding and dimerization, the ability of various RMF mutants to form 100S ribosomes was examined

Summary

Introduction

In the exponential growth phase of bacteria, several proteins are synthesized by a massive amount of active ribosomes through four stages: initiation, elongation, termination, and recycling (Korostelev et al, 2008). In Escherichia coli, the expression of RMF is positively regulated by the stringent response alarmone (p)ppGpp (guanosine-3 ,5 -bisdiphosphate or guanosine pentaphosphate) (Izutsu et al, 2001), the carbon source-sensing cAMP–cAMP receptor protein complex (cAMPCRP) (Shimada et al, 2013), and the transcription factors stimulating biofilm formation such as McbR, RcdA, and SdiA (Yoshida et al, 2018) Another protein factor expressed during the stationary phase, hibernation promoting factor (HPF, known as YhbH, Mr = 10,732), binds to ribosomes and promotes 100S ribosome formation (Maki et al, 2000; Ueta et al, 2005, 2008). We term the stage of inactivating ribosome “hibernation stage” in the ribosome cycle (Yoshida et al, 2002)

Methods

Results

Conclusion