Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape.
The Escherichia coli 30S ribosomal subunit self-assembles in vitro in a hierarchical manner, with the RNA binding by proteins enabled by the prior binding of others under equilibrium conditions. Early 16S rRNA binding proteins also bind faster than late-binding proteins, but the specific causes for the slow binding of late proteins remain unclear. Previously, a pulse-chase monitored by quantitative mass spectrometry method was developed for monitoring 30S subunit assembly kinetics, and here a modified experimental scheme was used to probe kinetic cooperativity by including a step where subsets of ribosomal proteins bind and initiate assembly prior to the pulse-chase kinetics. In this work, 30S ribosomal subunit kinetic reconstitution experiments revealed that thermodynamic dependency does not always correlate with kinetic cooperativity. Some folding transitions that cause subsequent protein binding to be more energetically favorable do not result in faster protein binding. Although 3(') domain primary protein S7 is required for RNA binding by both proteins S9 and S19, prior binding of S7 accelerates the binding of S9, but not S19, indicating there is an additional mechanistic step required for S19 to bind. Such data on kinetic cooperativity and the presence of multiphasic assembly kinetics reveal complexity in the assembly landscape that was previously hidden.
Bunner
Anne E
AE
Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, CA 92037, USA.
Beck
Andrea H
AH
Williamson
James R
JR
D055592:Biophysical Phenomena; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism; D054680:Ribosome Subunits, Small, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D012329:RNA, Bacterial; D012269:Ribosomal Proteins