Transient Protein-RNA Interactions Guide Nascent Ribosomal RNA Folding

Duss, Olivier; Stepanyuk, Galina A.; Puglisi, Joseph D.; Williamson, James R.

doi:10.1016/j.cell.2019.10.035

Cited by 74 publications

(75 citation statements)

References 72 publications

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“…1c, respectively). This may suggest that co-transcriptional folding of 16S rRNA in the presence of ribosomal proteins facilitated the assembly efficiency, as recently revealed 18,19 . Also note that a long-term analysis showed that R-iSAT continued for almost half a day with very slow kinetics ( Supplementary Fig.…”

Section: Resultsmentioning

confidence: 65%

In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E. coli

et al. 2020

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In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features. In this study, we integrated all of the processes required for Escherichia coli small ribosomal subunit assembly. In our method, termed fully Recombinant-based integrated Synthesis, Assembly, and Translation (R-iSAT), assembly and evaluation of the small ribosomal subunits are coupled with ribosomal RNA (rRNA) synthesis in a reconstituted cell-free protein synthesis system. By changing the components of R-iSAT, including recombinant ribosomal protein composition, we coupled ribosomal assembly with ribosomal protein synthesis, enabling functional synthesis of ribosomal proteins and subsequent subunit assembly. In addition, we assembled and evaluated subunits with mutations in both rRNA and ribosomal proteins. The study demonstrated that our scheme provides new ways to comprehensively analyze any elements of the small ribosomal subunit, with the goal of improving our understanding of ribosomal biogenesis, function, and engineering.

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Section: Resultsmentioning

confidence: 65%

In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E. coli

et al. 2020

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“…One implication is that cellular RNA helicases or even ribosomes can recommit the fate of these translational riboswitches long after transcription has completed. As many nucleic acid folding processes are kinetically controlled, including those involving ribonucleoprotein complexes 40 , 41 , we believe this method is broadly applicable to many types of RNA and even DNA structures, as it has been demonstrated in ribozymes (e.g., Twister 23 ), RNA aptamers (e.g., Spinach and Mango 24 ) and even telomeric DNA sequences in an experimental format that mimics co-reverse transcriptional telomeric DNA folding 36 , and to the studies of nucleic acid-protein complex assemblies.…”

Section: Discussionmentioning

confidence: 99%

Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

Hua¹,

Jones

Mitra

et al. 2020

Nat Commun

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RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5′-to-3′ direction, at ~60 nt s−1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.

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“…Ribosome assembly is an efficient but complicated process (Warner, 1999;Davis and Williamson, 2017). During this process, transient rRNA-RP interactions chaperone rRNA folding, which is essential for pre-rRNA processing and the following rRNA-RP assembly, and many other assembly factors have been identified (Bohnsack and Bohnsack, 2019;Duss et al, 2019;Rodgers et al, 2019;Prattes et al, 2019), indicating that assembly of ribosomes, especially heterogeneous ribosomes, is significantly more complex than previously thought. 4.…”

Section: Open Questionsmentioning

confidence: 99%

Ribosome heterogeneity in stem cells and development

Wang

2020

Journal of Cell Biology

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Translation control is critical to regulate protein expression. By directly adjusting protein levels, cells can quickly respond to dynamic transitions during stem cell differentiation and embryonic development. Ribosomes are multisubunit cellular assemblies that mediate translation. Previously seen as invariant machines with the same composition of components in all conditions, recent studies indicate that ribosomes are heterogeneous and that different ribosome types can preferentially translate specific subsets of mRNAs. Such heterogeneity and specialized translation functions are very important in stem cells and development, as they allow cells to quickly respond to stimuli through direct changes of protein abundance. In this review, we discuss ribosome heterogeneity that arises from multiple features of rRNAs, including rRNA variants and rRNA modifications, and ribosomal proteins, including their stoichiometry, compositions, paralogues, and posttranslational modifications. We also discuss alterations of ribosome-associated proteins (RAPs), with a particular focus on their consequent specialized translational control in stem cells and development.

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Transient Protein-RNA Interactions Guide Nascent Ribosomal RNA Folding

Cited by 74 publications

References 72 publications

In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E. coli

In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E. coli

Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

Ribosome heterogeneity in stem cells and development

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