Structural Studies Reveal the Role of Helix 68 in the Elongation Step of Protein Biosynthesis

Cimicata, Giuseppe; Fridkin, G.; Bose, Tanaya; Eyal, Zohar; Halfon, Y.; Breiner-Goldstein, Elinor; Fox, Tara; Zimmerman, Ella; Bashan, Anat; Val, Natalia de; Wlodawer, Alexander; Yonath, Ada

doi:10.1128/mbio.00306-22

Cited by 8 publications

(6 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Clearly, a dynamic equilibrium exists in H67-H71 conformations of the log phase 50S subunits. Interestingly, dynamic nature of H68 has also been reported in recent study of S. aureus 50S subunit, where a ∼70° displacement of H68 from its usual position towards L1 stalk is seen (Cimicata et al, 2022). Notably, 3D structures prediction of mycobacterial H67-H71 by RNAComposer web server, resulted in a curved conformation of H68 of mycobacterium 23S rRNA (Fig.…”

Section: Resultssupporting

confidence: 63%

Cryo-EM structures of the Mycobacterium 50S subunit reveal an intrinsic conformational dynamics of 23S rRNA helices

Baid

Sengupta

2022

Preprint

View full text Add to dashboard Cite

Pathogenic organisms encounter a broad range of stress conditions within host micro-environment and adopt variety of mechanisms to stall protein translation and protect translational machinery. Structural investigations of the ribosomes isolated from pathogenic and non-pathogenic Mycobacterium species have identified several mycobacteria-specific structural features of ribosomal RNA and proteins. Here, we report a growth phase-dependent conformational switch of domain III and IV helices (H54a and H67-H71) of the mycobacterium 23S rRNA. Cryo-electron microscopy (cryo-EM) structures (~3-4 Å) of the M. smegmatis (Msm) 50S ribosomal subunit of log-phase manifested that, while H68 possesses the usual stretched conformation in one of the maps, another one exhibits an unprecedented conformation of H68 curling onto a differently oriented H69, indicating an intrinsic dynamic nature of H68. Remarkably, a 2.8Å cryo-EM map of the Msm stationary-state 50S subunit unveiled that H68 preferably acquires folded conformation in this state (closely mimicking dormant state). Formation of a bulge-out structure by H68 at the inter-subunit surface of the stationary-state 50S subunit due to the rRNA conformational changes prevents association with 30S subunit and keeps an inactive pool of the 50S subunit representing a ribosome-protection mechanism during dormancy. Evidently, this dynamic nature of H68 is an integral part of the cellular functions of mycobacterium ribosome, and irreversibly arresting H68 flexible motion would stall ribosome function. Thus, this conformational change may be exploited to develop anti-mycobacterium drug molecules.

show abstract

Section: Resultssupporting

confidence: 63%

Cryo-EM structures of the Mycobacterium 50S subunit reveal an intrinsic conformational dynamics of 23S rRNA helices

Baid

Sengupta

2022

Preprint

View full text Add to dashboard Cite

show abstract

“…This disorder, also present for the usually flexible H69, seems to be greater than the alternate conformations identified for H68 in the Sau 50S subunit and 70S ribosome at physiological temperature (Supplementary Fig. 13 , Supplementary movie 4 ) that were suggested to be involved in the ribosomal translocation mechanism 47 . A lowering of the density threshold values shows some ordering of H68 but only with simultaneous appearance of the fragmented 30S subunit densities, suggesting that this ordering may be attributable to the small number of 70S particles in this class, which could not be computationally removed.…”

Section: Resultsmentioning

confidence: 76%

The structure of a hibernating ribosome in a Lyme disease pathogen

Sharma,

Manjari,

Agrawal

et al. 2023

Nat Commun

View full text Add to dashboard Cite

The spirochete bacterial pathogen Borrelia (Borreliella) burgdorferi (Bbu) affects more than 10% of the world population and causes Lyme disease in about half a million people in the US annually. Therapy for Lyme disease includes antibiotics that target the Bbu ribosome. Here we present the structure of the Bbu 70S ribosome obtained by single particle cryo-electron microscopy at 2.9 Å resolution, revealing a bound hibernation promotion factor protein and two genetically non-annotated ribosomal proteins bS22 and bL38. The ribosomal protein uL30 in Bbu has an N-terminal α-helical extension, partly resembling the mycobacterial bL37 protein, suggesting evolution of bL37 and a shorter uL30 from a longer uL30 protein. Its analogy to proteins uL30m and mL63 in mammalian mitochondrial ribosomes also suggests a plausible evolutionary pathway for expansion of protein content in mammalian mitochondrial ribosomes. Computational binding free energy predictions for antibiotics reflect subtle distinctions in antibiotic-binding sites in the Bbu ribosome. Discovery of these features in the Bbu ribosome may enable better ribosome-targeted antibiotic design for Lyme disease treatment.

show abstract

“…For example, the 23S rRNA sequences of operons A, C, E, and H differ from that of B, D, and G at Helix 98 (H98), a motif that has been shown to participate in a tertiary interaction that is important for ribosome stabilization . Similarly, H68, a 23S rRNA helix that is extended by a Watson–Crick (WC) base pair in the A operon is known to be actively involved in dynamic ribosome movements that are necessary for the process of elongation via coordination with the L1 stalk and tRNAs . The additional WC base pair, which would increase the length of H68, potentially affects the dynamics of the interaction with the L1 stalk, altering the efficiency of the elongation process.…”

Section: Resultsmentioning

confidence: 99%

“…29 Similarly, H68, a 23S rRNA helix that is extended by a Watson−Crick (WC) base pair in the A operon is known to be actively involved in dynamic ribosome movements that are necessary for the process of elongation via coordination with the L1 stalk and tRNAs. 30 The additional WC base pair, which would increase the length of H68, potentially affects the dynamics of the interaction with the L1 stalk, altering the efficiency of the elongation process. In the 16S rRNA, operon H carries seven unique polymorphisms in helix 33 (h33), which sits in the "head" of the small subunit and plays an important role in ratcheting along the mRNA in the process of translation.…”

Section: Activity Of Ribosomes Derived From Single Rrnamentioning

confidence: 99%

Ribosome Pool Engineering Increases Protein Biosynthesis Yields

Kofman,

Willi,

Karim

et al. 2024

ACS Cent. Sci.

View full text Add to dashboard Cite

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.

show abstract

Structural Studies Reveal the Role of Helix 68 in the Elongation Step of Protein Biosynthesis

Cited by 8 publications

References 46 publications

Cryo-EM structures of the Mycobacterium 50S subunit reveal an intrinsic conformational dynamics of 23S rRNA helices

Cryo-EM structures of the Mycobacterium 50S subunit reveal an intrinsic conformational dynamics of 23S rRNA helices

The structure of a hibernating ribosome in a Lyme disease pathogen

Ribosome Pool Engineering Increases Protein Biosynthesis Yields

Contact Info

Product

Resources

About