Ribosomes are complex macromolecular machines that translate the genetic code. Over the past decade, numerous insights into the process of translation have been gained through structural studies of ribosomes using x-ray crystallography. However, a limiting factor in the study of ribosome dynamics and functional interactions is the ability to produce crystals of ribosomes in alternative arrangements or in complex with additional factors.
For the purpose of crystallizing recalcitrant ribosome complexes, I conducted a phylogenetic screen for new crystal forms. Species were selected from environmental samples for superior growth and behavior in a laboratory setting, and new ribosome crystals were obtained using naturally inhibited ribosomes from dormant bacteria. The structure of the translational inhibitor ribosome modulation factor (RMF) bound to the E. coli ribosome shows how this small protein is able to inhibit translation and protect ribosomes from degradation by binding to the intersubunit cavity.
One of the most successful species to come from the crystal screen was the gram-positive bacterium Planococcus donghaensis strain MPA1U2. The dormant 100S ribosome dimers from this phychrotroph were structurally investigated using a combination of electron microscopy and x-ray crystallography and found to have a unique stable interaction between 70S ribosomes. The P. donghaensis genome sequence was determined and reveals that this organism has eliminated spore formation, but retained its master regulator as part of its stress response pathway.
Crystallization of ribosomes has allowed detailed structural information to be obtained about the mechanism of translation. The unique properties of ribosome crystals could also be used to obtain structural information about other ribonucleoprotein complexes by tethering them together genetically. I have made significant strides toward this goal by crystallizing ribosomes containing RNA insertions in external loops of the rRNA.
|Advisor:||Noller, Harry F.|
|School:||University of California, Santa Cruz|
|School Location:||United States -- California|
|Source:||DAI-B 72/06, Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology, Biochemistry|
|Keywords:||Crystal forms, Crystal structures, Dormant bacteria, Polygenetic screen, Ribosome modulation factors, Ribosomes|
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