Proteins, the workhorse of a biological cell, are produced and distributed by a large, flexible, macromolecular machine called the ribosome. Messenger RNA (mRNA) is translated by the ribosome to create a protein in a process that involves molecular movements of the ribosome and determining the various structural conformations involved can provide insight toward understanding ribosomal function. Currently, the field of functionally oriented structural biology emphasizes the importance of mapping conformational space. However, mapping the continuous structural variation of the ribosome is difficult due to low energetic barriers between ribosomal conformational states. This difficulty has interfered in characterizing the conformational distortions of the 70S E. coli ribosome as it performs a helicase mechanism to unwind structured mRNA, an important phenomenon that impacts the production and diversity of bacterial proteome. This study uses experimental small-angle scattering and molecular modeling methods to divulge the structural dimensions of 70S E. coli ribosome in solution.
Contrast variation small-angle neutron scattering was used to distinguish the scattering contribution of the two components, rRNA and ribosomal proteins, that comprise the ribosome. We compared the observed 70S ribosomes to eleven all-atom structural models derived from X-ray crystallography and cryo-EM structures. The E. coli 70S bacterial ribosome is made up of three significant RNA components that make up 2/3 of the mass and 54 small ribosomal proteins (r-proteins) that contribute 1/3 of the mass of the complex. The rRNA makes up the core, and the r-proteins are dotted around the periphery of the complex. rRNA extends by about ~10% and r-protein are ~35% more extended in solution compared to ribosomes from x-ray crystallography and cryo-EM techniques. During helicase activity r-proteins elongate by 10% and rRNA does not have detectible structural changes, indicating that r-proteins play a notable role in helicase activity. This work has two significant impacts, it shows novel flexibility of the 70S E. coli ribosome and provides evidence that r-proteins make meaningful contributions to specialized tasks of a ribosome, such as the E.coli ribosome’s task of producing a diverse proteome from a small bacterial genome.
Furthermore, unique insights offered in this thesis compares ribosomes while flexible in liquid using two microscopy techniques. The solution structure was found using SAS, as discussed above, which found the ribosomal proteins are flexible and can be highly extended. The less discussed highlight of this work is the imaging of the ribosome using tapping mode AFM in liquid. Similarities in these sample environments and in the ribosome purification technique allow for a nearly direct comparison of ribosome structure in solution using two ”lenses”. One lens observes the ribosomes properties as a polymer intercalated with liquid (SAS); the other observes the ribosomes properties as a spring on a surface (AFM).
|Advisor:||Cornish, Peter V|
|Commitee:||Burke, Donald H, Kaiser, Helmut, King, Gavin, Jiji, Renee|
|School:||University of Missouri - Columbia|
|School Location:||United States -- Missouri|
|Source:||DAI-B 81/10(E), Dissertation Abstracts International|
|Subjects:||Biophysics, Biochemistry, Biology|
|Keywords:||70S, Contrast variation, DnaX, Helicase, Ribosome, Small angle scattering|
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