Interactions between terminal ribosomal RNA helices stabilize the E. coli large ribosomal subunit

Nissley, Amos J.; Kamal, Tammam S.; Cate, Jamie H. D.

doi:10.1261/rna.079690.123

Interactions between terminal ribosomal RNA helices stabilize the E. coli large ribosomal subunit

Journal Article · Fri Jul 07 00:00:00 EDT 2023 · RNA

DOI:https://doi.org/10.1261/rna.079690.123· OSTI ID:2470936

^[1]; Kamal, Tammam S. ^[1]; ^[2]

Univ. of California, Berkeley, CA (United States)
Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding. In vivo assembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and function in vitro. RNA tags in the Escherichia coli 50S subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth or in vitro ribosome activity. Here, we find that E. coli 50S subunits with MS2 tags inserted in H98 are destabilized compared to wild-type (WT) 50S subunits. We identify the loss of RNA–RNA tertiary contacts that bridge helices H1, H94, and H98 as the cause of destabilization. Using cryogenic electron microscopy (cryo-EM), we show that this interaction is disrupted by the addition of the MS2 tag and can be restored through the insertion of a single adenosine in the extended H98 helix. This work establishes ways to improve MS2 tags in the 50S subunit that maintain ribosome stability and investigates a complex RNA tertiary structure that may be important for stability in various bacterial ribosomes.

View Accepted Manuscript (DOE)

Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: National Science Foundation (NSF); USDOE

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 2470936

Journal Information:: RNA, Journal Name: RNA Journal Issue: 10 Vol. 29; ISSN 1355-8382

Publisher:: Cold Spring Harbor Laboratory PressCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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Interactions between terminal ribosomal RNA helices stabilize the E. coli large ribosomal subunit

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