Kinetic cooperativity in Escherichia coli 30S ribosomal subunit reconstitution reveals additional complexity in the assembly landscape

被引:39
作者
Bunner, Anne E. [1 ,2 ,3 ]
Beck, Andrea H. [1 ,2 ,3 ]
Williamson, James R. [1 ,2 ,3 ]
机构
[1] Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA
[2] Scripps Res Inst, Dept Chem, La Jolla, CA 92037 USA
[3] Scripps Res Inst, Skaggs Inst Chem Biol, La Jolla, CA 92037 USA
基金
美国国家卫生研究院;
关键词
mass spectrometry; RNA folding; RNA-protein interactions; IN-VIVO; RNA; PROTEINS; BIOGENESIS; DOMAIN;
D O I
10.1073/pnas.0912007107
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:5417 / 5422
页数:6
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