Protein-depleted ribosomes as a means for selective translation of conditionally leaderless mRNAs under stress conditions

One of the most intricate and fundamental processes of life is the translation of the genetic code into proteins. Decoding the mRNA-based information into the corresponding sequence of amino acids is performed by a complex ribonucleoprotein particle, the ribosome. Due to its structural and functional complexity the ribosome represents a major target for antibiotics. The aminoglycoside antibiotic kasugamycin (Ksg) inhibits translation at the step of initiation by blocking the mRNA path on the 30S ribosomal subunit (Schluenzen et al., 2006). However, translation of leaderless mRNAs, which start directly with a 5´-terminal AUG and lack kingdom-specific ribosome recruitment signals, prevails in E. coli in the presence of the drug in vivo (Moll and Bläsi, 2002). Our studies revealed that the presence of the antibiotic results in the formation of ribosomes depleted for several essential proteins of the small subunit including the functionally important proteins S1 and S12. However, these 61S ribosomes are proficient in translation of leaderless mRNAs in vivo and in vitro. These studies provided therefore the first evidence for the functionality of protein-deficient ribosomes in translation and supported the hypothesis, that the modern ribosome is a protein-stabilized ribozyme. In addition, we were able to reconcile the lack of these proteins with structural rearrangements in the 16S rRNA upon binding of Ksg to 70S ribosomes, which enabled us to devise a model for the formation of these particles originating from fully assembled 70S ribosomes (Kaberdina et al., 2009). As leaderless mRNAs are translated by ribosomes from all three domains of life, they are suggested to represent remnants of ancestral mRNAs. Therefore, these studies support in addition the hypothesis that the “61S” particles might reflect an intermediate step of ribosome evolution before the kingdoms have diverged. 
During these studies we observed the resumed synthesis of specific proteins upon prolonged Ksg treatment. We identified some of these proteins as chaperones, stress proteins, ribosomal proteins and ribosome modifying enzymes. Surprisingly, we discovered that the respective mRNAs became leaderless in the presence of the antibiotic, which apparently allows translation by protein-depleted ribosomes. Taken together, these results suggest that translation of conditionally leaderless mRNAs by protein-depleted ribosomes might represent a novel regulatory mechanism under adverse/stress conditions. Therefore, the main focus of our group is the determination of the molecular mechanism(s) leading to the 5´-terminal processing of the respective mRNAs and the formation of protein depleted ribosomes under stress conditions.

The interaction between ribosomal proteins S1 and S2: A target for novel antimicrobials against Gram-negative pathogens?

Protein S1 with a molecular mass of 61kD is associated with the small subunit of the ribosome and represents the largest of all Escherichia coli ribosomal proteins. It is one of the few ribosomal proteins to which a function has been assigned. It has been implicated in translation initiation by binding to a region rich in pyrimidines upstream of the SD-sequence, thereby increasing the concentration of the translational start site in the vicinity of the decoding site on the ribosome. Therefore, the protein is pivotal for translation initiation in E. coli and all Gram-negative bacteria studied so far. Although the structure of the ribosome is solved at atomic resolution, the position of protein S1 on the ribosome is still unknown, due to its intrinsic flexibility. In previous studies, we were able to show that ribosomal protein S2 is absolutely required for binding of S1 to the 30S ribosomal subunit (Moll et al., 2002). Therefore, the second aim of our group is to characterize the interaction between proteins S1 and S2 to shed light on the binding mode of S1 to the ribosome. Besides this essential contribution to basic research, we are interested to identify a chemical compound with the potential to interfere with this interaction. Such a compound could have the potential to be used as a semi-selective antimicrobial against Gram-negative pathogens, including Pseudomonas aeruginosa, Salmonella enterica, Escherichia coli and Klebsiella pneumoniae, without affecting the majority of the beneficial Gram-positive flora, as many of them do not possess a homologue of S1. As mitochondrial ribosomes lack functional homologues of S1 and eukaryotic ribosomes employ different mechanisms during translation initiation, a drug, which specifically inhibits binding of protein S1 to the bacterial ribosome, is not expected to interfere with protein synthesis in eukaryotic cells or organelles, and should therefore not exhibit toxic side effects.
 
 
 
 
References
 
Kaberdina AC, Szaflarski W, Nierhaus KH, Moll I (2009) An unexpected type of ribosomes induced by kasugamycin: a look into ancestral times of protein synthesis? Mol Cell 33(2): 227-236
 
Moll I, Blasi U (2002) Differential inhibition of 30S and 70S translation initiation complexes on leaderless mRNA by kasugamycin. Biochem Biophys Res Commun 297(4): 1021-1026
 
Moll I, Grill S, Grundling A, Blasi U (2002) Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD-box helicase on translation of leaderless and canonical mRNAs in Escherichia coli. Mol Microbiol 44(5): 1387-1396
 
Schluenzen F, Takemoto C, Wilson DN, Kaminishi T, Harms JM, Hanawa-Suetsugu K, Szaflarski W, Kawazoe M, Shirouzo M, Nierhaus KH, Yokoyama S, Fucini P (2006) The antibiotic kasugamycin mimics mRNA nucleotides to destabilize tRNA binding and inhibit canonical translation initiation. Nature Structural & Molecular Biology 13(10): 871-878