Heteronuclear NMR investigations of dynamic regions of intact Escherichia coli ribosomes

Abstract

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...

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

“…Some of the more successful approaches for characterizing ribosomes include crosslinking [1][2][3][4], cryo-electron microscopy (cryo-EM) [5][6][7], tritium bombardment [8], nuclear magnetic resonance (NMR) [9] and electrospray mass spectrometry [10][11][12][13]. One of the most successful approaches has been the development of X-ray crystallography within the past several years, wherein high-resolution crystal structures of ribosomal subunits or intact ribosomes have been obtained that provide detailed structural information at the atomic level [14][15][16][17][18].…”

Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy

“…By contrast, the values expected for the 70S ribosome particle are Ͼ1,000 Hz (24,25). The addition of 70S ribosomes to purified Ig2 is done to account for the nonspecific increases in sample viscosity and crowding that are associated with the presence of ribosomes and is a similar control to that used in our previous NMR study of the E. coli ribosome (20).…”

“…This size suggests that both the resonance linewidths and the complexity of the spectra would be far too great for NMR to be used to study the properties of any nascent chain attached to such a complex. Nonetheless, we and others have shown (20,21) that a number of well resolved resonances can be observed in NMR spectra of the ribosome itself as a result of the independent motion of localized regions of the structure. This has enabled us to define the structure and dynamics of the L7/L12 proteins in the mobile GTPase-associated region (GAR or stalk region) of the E. coli ribosome that is involved in the regulation of protein elongation (19).…”

Protein structure determination from NMR chemical shifts

“…This approach should enable a description of the structural and dynamical properties of specific proteins under a variety of conditions to be obtained. Furthermore, recent studies are showing that new approaches can permit NMR spectra to be obtained and assigned for systems that have previously appeared inaccessible to this spectroscopic technique, including large or transient multimolecular assemblies (12,14,15), low-populated states involved in enzymatic catalysis, allosteric communication, and protein folding (7,8), and proteins associated with membranes (13). The ability to define detailed structures from chemical shifts by using the type of approach described in the present study could be crucial in addressing the structural challenges associated with such systems and hence play an increasingly important and unique role in structural and molecular biology.…”

“…It has also been recognized that chemical shifts can aid in the determination of the tertiary structure of proteins when used in combination with other NMR probes that report on interproton distances (NOEs) and the relative orientations of the different nuclei in a protein structure [residual dipolar couplings (RDC)] (3,6,10). In many important cases, however, chemical shifts are the only NMR parameters that can be obtained on a given state of a protein with any degree of completeness (7,8,(11)(12)(13)(14)(15), prompting us to explore the extent to which these quantities alone can be used to determine high-resolution structures.…”