An assembly landscape for the 30S ribosomal subunit.
Self-assembling macromolecular machines drive fundamental cellular processes, including transcription, messenger RNA processing, translation, DNA replication and cellular transport. The ribosome, which carries out protein synthesis, is one such machine, and the 30S subunit of the bacterial ribosome is the preeminent model system for biophysical analysis of large RNA-protein complexes. Our understanding of 30S assembly is incomplete, owing to the challenges of monitoring the association of many components simultaneously. Here we have developed a method involving pulse-chase monitored by quantitative mass spectrometry (PC/QMS) to follow the assembly of the 20 ribosomal proteins with 16S ribosomal RNA during formation of the functional particle. These data represent a detailed and quantitative kinetic characterization of the assembly of a large multicomponent macromolecular complex. By measuring the protein binding rates at a range of temperatures, we find that local transformations throughout the assembling subunit have similar but distinct activation energies. Thus, the prevailing view of 30S assembly as a pathway proceeding through a global rate-limiting conformational change must give way to one in which the assembly of the complex traverses a landscape dotted with various local conformational transitions.
Talkington
Megan W T
MW
Department of Molecular Biology, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Siuzdak
Gary
G
Williamson
James R
JR
D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012336:RNA, Ribosomal, 16S / Q000235:genetics / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D016963:Thermus thermophilus
D021122:Protein Subunits; D012336:RNA, Ribosomal, 16S; D012269:Ribosomal Proteins