Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome

Nissley, Amos J; Penev, Petar I.; Watson, Zoe L; Banfield, Jillian F.; Cate, Jamie H. D.

doi:10.1093/nar/gkac1273

Cited by 8 publications

(28 citation statements)

References 73 publications

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“…Mutant E. coli 50S ribosomal subunits were expressed and purified as previously described (14). Briefly, E. coli NEB Express I q cells were transformed with the corresponding pLK35 plasmid.…”

Section: Methodsmentioning

confidence: 99%

“…tRNA fMet with a C1G mutation and a modified terminal 3′-NH2-ATP was in vitro transcribed and modified as previously described (14). fMet-tRNA fMet was prepared enzymatically as described (18).…”

Section: Fmet-trna Fmet Preparationmentioning

confidence: 99%

“…Junk particles were rejected and a second round of 2D classification was performed to further remove the remaining junk particles. Selected particles were re-extracted and Fourier-cropped to 1/4 of the box size and 3D classification was performed with heterogeneous refinement in cryoSPARC using a 70S ribosome map from (14). Classes containing features consistent with 70S ribosomes were combined and subjected to another round of 3D classification.…”

Section: Cryo-em Data Acquisition Image Processing and Modelingmentioning

confidence: 99%

“…We examined the H1-H94-H98 interaction in the cryo-EM structures of a WT 70S ribosome (19) and ribosomes with an MS2-V2 tag (14). In the WT ribosome, there is clear density for G9 (H1), A10 (H1), and U2629 (H94) sugars and bases.…”

Section: Ms2-v3 Restores Stability To the E Coli Lsumentioning

confidence: 99%

“…MS2 tags inserted in H98 of 23S rRNA have been used extensively to enable the investigation of rRNA function (10, 11), in engineered ribosomes (12), and for in vivo ribosome tracking (13). Although E. coli 50S ribosomal subunits with MS2 tags inserted into H98 retain wild-type (WT) in vivo and in vitro function, we previously showed that these 50S subunits have lower thermostability compared to WT 50S subunits (14).…”

Section: Introductionmentioning

confidence: 99%

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Interactions between terminal ribosomal RNA helices stabilize theEscherichia colilarge ribosomal subunit

Nissley

Kamal

Cate

2023

Preprint

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The ribosome is a large ribonucleoprotein assembly that uses diverse and complex molecular interactions to maintain proper folding.In vivoassembled ribosomes have been isolated using MS2 tags installed in either the 16S or 23S ribosomal RNAs (rRNAs), to enable studies of ribosome structure and functionin vitro. RNA tags in theEscherichia colilarge ribosomal (50S) subunit have commonly been inserted into an extended helix H98 in 23S rRNA, as this addition does not affect cellular growth orin vitroribosome activity. Here, we find thatE. coli50S 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.

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Section: Methodsmentioning

confidence: 99%

Section: Fmet-trna Fmet Preparationmentioning

confidence: 99%

Section: Cryo-em Data Acquisition Image Processing and Modelingmentioning

confidence: 99%

Section: Ms2-v3 Restores Stability To the E Coli Lsumentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Interactions between terminal ribosomal RNA helices stabilize theEscherichia colilarge ribosomal subunit

Nissley

Kamal

Cate

2023

Preprint

Self Cite

View full text Add to dashboard Cite

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Ribosome Pool Engineering Increases Protein Biosynthesis Yields

Kofman,

Willi,

Karim

et al. 2024

ACS Cent. Sci.

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The biosynthetic capability of the bacterial ribosome motivates efforts to understand and harness sequenceoptimized versions for synthetic biology. However, functional differences between natively occurring ribosomal RNA (rRNA) operon sequences remain poorly characterized. Here, we use an in vitro ribosome synthesis and translation platform to measure protein production capabilities of ribosomes derived from all unique combinations of 16S and 23S rRNAs from seven distinct Escherichia coli rRNA operon sequences. We observe that polymorphisms that distinguish native E. coli rRNA operons lead to significant functional changes in the resulting ribosomes, ranging from negligible or low gene expression to matching the protein production activity of the standard rRNA operon B sequence. We go on to generate strains expressing single rRNA operons and show that not only do some purified in vivo expressed homogeneous ribosome pools outperform the wild-type, heterogeneous ribosome pool but also that a crude cell lysate made from the strain expressing only operon A ribosomes shows significant yield increases for a panel of medically and industrially relevant proteins. We anticipate that ribosome pool engineering can be applied as a tool to increase yields across many protein biomanufacturing systems, as well as improve basic understanding of ribosome heterogeneity and evolution.

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Costs of ribosomal RNA stabilization affect ribosome composition at maximum growth rate

Széliová,

Müller,

Zanghellini

2024

Commun Biol

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Ribosomes are key to cellular self-fabrication and limit growth rate. While most enzymes are proteins, ribosomes consist of 1/3 protein and 2/3 ribonucleic acid (RNA) (in E. coli).Here, we develop a mechanistic model of a self-fabricating cell, validated across diverse growth conditions. Through resource balance analysis (RBA), we explore the variation in maximum growth rate with ribosome composition, assuming constant kinetic parameters.Our model highlights the importance of RNA instability. If we neglect it, RNA synthesis is always cheaper than protein synthesis, leading to an RNA-only ribosome at maximum growth rate. Upon accounting for RNA turnover, we find that a mixed ribosome composed of RNA and proteins maximizes growth rate. To account for RNA turnover, we explore two scenarios regarding the activity of RNases. In (a) degradation is proportional to RNA content. In (b) ribosomal proteins cooperatively mitigate RNA instability by protecting it from misfolding and subsequent degradation. In both cases, higher protein content elevates protein synthesis costs and simultaneously lowers RNA turnover expenses, resulting in mixed RNA-protein ribosomes. Only scenario (b) aligns qualitatively with experimental data across varied growth conditions.Our research provides fresh insights into ribosome biogenesis and evolution, paving the way for understanding protein-rich ribosomes in archaea and mitochondria.

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Rare ribosomal RNA sequences from archaea stabilize the bacterial ribosome

Cited by 8 publications

References 73 publications

Interactions between terminal ribosomal RNA helices stabilize theEscherichia colilarge ribosomal subunit

Interactions between terminal ribosomal RNA helices stabilize theEscherichia colilarge ribosomal subunit

Ribosome Pool Engineering Increases Protein Biosynthesis Yields

Costs of ribosomal RNA stabilization affect ribosome composition at maximum growth rate

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