Abstract
Post-transcriptional ribosomal RNA (rRNA) modifications are present in all organisms, but their exact functional roles and positions are yet to be fully characterized. Modified nucleotides have been implicated in the stabilization of RNA structure and regulation of ribosome biogenesis and protein synthesis. In some instances, rRNA modifications can confer antibiotic resistance. High-resolution ribosome structures are thus necessary for precise determination of modified nucleotides’ positions, a task that has previously been accomplished by X-ray crystallography. Here, we present a cryo-electron microscopy (cryo-EM) structure of the Escherichia coli 50S subunit at an average resolution of 2.2 Å as an additional approach for mapping modification sites. Our structure confirms known modifications present in 23S rRNA and additionally allows for localization of Mg2+ ions and their coordinated water molecules. Using our cryo-EM structure as a testbed, we developed a program for assessment of cryo-EM map quality. This program can be easily used on any RNA-containing cryo-EM structure, and an associated Coot plugin allows for visualization of validated modifications, making it highly accessible.
Highlights
Ribosomes are complex cellular machines responsible for protein synthesis
Using a Titan Krios X-FEG instrument equipped with a K2 direct electron detector camera operated in super-resolution mode and performing reconstruction refinement in Relion3, CryoSPARC and cisTEM software, we obtained a structure of the E. coli 50S subunit at an average resolution of 2.2 A (Table 1 and Figure 1)
We present a new program for assessing map quality, named qPTxM, which demonstrates the superior quality of the present map
Summary
Ribosomes are complex cellular machines responsible for protein synthesis. Accurate translation by the ribosome requires specific post-transcriptional and post-translational modifications of ribosomal RNA (rRNA) and ribosomal proteins, respectively. The roles of individual rRNA modifications are still poorly understood, and while some modulate the function of the ribosome, others are important in RNA folding and stability [1,2,3,4]. Differential expression and posttranslational modifications of ribosomal proteins, as well as differences in rRNA modifications, can lead to heterogeneity in ribosome composition which can result in ‘specialized ribosomes’. These ribosomes can have a substantial impact on the relative abundances of proteins being produced and influence an organism’s adaptability to a variety of environmental factors [9,10,11]. In bacteria, ribosome heterogeneity has been identified as a mechanism for stress adaptation [12]
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