15 N-1 H NMR spectroscopy has been used to probe the dynamic properties of uniformly 15 N labeled Escherichia coli ribosomes. Despite the high molecular weight of the complex (Ϸ2.3 MDa), [ 1 H-15 N] heteronuclear single-quantum correlation spectra contain Ϸ100 well resolved resonances, the majority of which arise from two of the four C-terminal domains of the stalk proteins, L7͞L12. Heteronuclear pulse-field gradient NMR experiments show that the resonances arise from species with a translational diffusion constant consistent with that of the intact ribosome. Longitudinal relaxation time (T1) and T1 15 N-spin relaxation measurements show that the observable domains tumble anisotropically, with an apparent rotational correlation time significantly longer than that expected for a free L7͞L12 domain but much shorter than expected for a protein rigidly incorporated within the ribosomal particle. The relaxation data allow the ribosomally bound C-terminal domains to be oriented relative to the rotational diffusion tensor. Binding of elongation factor G to the ribosome results in the disappearance of the resonances of the L7͞L12 domains, indicating a dramatic reduction in their mobility. This result is in agreement with cryoelectron microscopy studies showing that the ribosomal stalk assumes a single rigid orientation upon elongation factor G binding. As well as providing information about the dynamical properties of L7͞L12, these results demonstrate the utility of heteronuclear NMR in the study of mobile regions of large biological complexes and form the basis for further NMR studies of functional ribosomal complexes in the context of protein synthesis. P rotein synthesis in living systems takes place on the ribosome, a complex macromolecular assembly whose structural and functional properties are rapidly emerging from a powerful combination of electron microscopy (EM) and x-ray crystallography (1, 2). In Escherichia coli, the ribosome is composed of 54 different proteins and three RNA molecules (23S, 16S, and 5S rRNA) This 2.3-MDa complex is termed the 70S ribosome and is made up of two components, the 30S and 50S subunits. The translation of genetic information into functional proteins involves a number of auxiliary factors, many of which are GTPases, including IF2, EF-Tu, elongation factor G (EF-G), and RF3 (2). These molecules bind to overlapping sites on the 50S subunit and regulate the transition of the ribosome through various states on the translational pathway. The binding sites are collectively known as the GTPase-associated region, due to the role of this region in stimulating the GTPase activity of the auxiliary factors.The GTPase-associated region (GAR) includes helices 42-44 and 95 (the ␣-sarcin loop) of 23S RNA, and the proteins L10, L11, and L7͞L12 (1). The latter (L7͞L12) is located on the ribosomal stalk and is unique among the ribosomal proteins, because it is the only protein present in multiple copies (four proteins per ribosomal particle). Although atomic details of much of the GAR have been reve...
We used mass spectrometry to identify proteins that are released in the gas phase from Escherichia coli ribosomes in response to a range of different solution conditions and cofactor binding. From solution at neutral pH the spectra are dominated by just 4 of the 54 ribosomal proteins (L7/L12, L11, and L10). Lowering the pH of the solution leads to the gas phase dissociation of four additional proteins as well as the 5 S RNA. Replacement of Mg 2؉ by Li ؉ ions in solutions of ribosomes induced the dissociation of 17 ribosomal proteins. Correlation of these results with available structural information for ribosomes revealed that a relatively high interaction surface area of the protein with RNA was the major force in preventing dissociation. By using the proteins that dissociate to probe their interactions with RNA, we examined different complexes of the ribosome formed with the elongation factor G and inhibited by fusidic acid or thiostrepton. Mass spectra recorded for the fusidic acid-inhibited complex reveal subtle changes in peak intensity of the proteins that dissociate. By contrast gas phase dissociation from the thiostrepton-inhibited complex is markedly different and demonstrates the presence of L5 and L18, two proteins that interact exclusively with the 5 S RNA. These results allow us to propose that the ribosome elongation factor-G complex inhibited by thiostrepton, but not fusidic acid, involves destabilization of 5 S RNA-protein interactions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.