{"id":1195,"date":"2024-01-21T12:00:00","date_gmt":"2024-01-21T10:00:00","guid":{"rendered":"https:\/\/sisu.ut.ee\/ribolab\/?p=1195"},"modified":"2024-10-23T11:42:50","modified_gmt":"2024-10-23T08:42:50","slug":"translational-impacts-of-enzymes-that-modify-ribosomal-rna-around-the-peptidyl-transferase-centre","status":"publish","type":"post","link":"https:\/\/sisu.ut.ee\/ribolab\/translational-impacts-of-enzymes-that-modify-ribosomal-rna-around-the-peptidyl-transferase-centre\/","title":{"rendered":"Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre"},"content":{"rendered":"<div class=\"wp-block-group is-layout-constrained wp-block-group-is-layout-constrained\">\n<p>Bao L, Liljeruhm J, Crespo Blanco R, Brandis G, Remme J, Forster AC. <\/p>\n\n\n\n<p>Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on\u00a0<em>Escherichia coli<\/em>\u00a0growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the \u2018critical region\u2019 of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37\u00b0C (although major at 20\u00b0C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20\u00b0C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains.\u00a0<em>In vitro<\/em>\u00a0fast kinetics at 20\u00b0C and 37\u00b0C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis\u00a0<em>in vivo<\/em>, as judged by the kinetics of \u03b2-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37\u00b0C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg<sup>2+<\/sup>. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications\u00a0<em>per se<\/em>, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.<\/p>\n\n\n\n<p>RNA Biol. 2024 Jan;21(1):31-41. doi: <a href=\"https:\/\/www.tandfonline.com\/doi\/full\/10.1080\/15476286.2024.2368305\" data-type=\"link\" data-id=\"https:\/\/www.tandfonline.com\/doi\/full\/10.1080\/15476286.2024.2368305\" target=\"_blank\" rel=\"noreferrer noopener\">10.1080\/15476286.2024.2368305.<\/a><br><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/38952121\/\" data-type=\"link\" data-id=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/38952121\/\" target=\"_blank\" rel=\"noreferrer noopener\">Pubmed<\/a><\/p>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Bao L, Liljeruhm J, Crespo Blanco R, Brandis G, Remme J, Forster AC. Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on\u00a0Escherichia coli\u00a0growth. Furthermore, &#8230;<\/p>\n","protected":false},"author":194,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"inline_featured_image":false,"footnotes":""},"categories":[11],"tags":[],"class_list":["post-1195","post","type-post","status-publish","format-standard","hentry","category-artiklid"],"acf":[],"_links":{"self":[{"href":"https:\/\/sisu.ut.ee\/ribolab\/wp-json\/wp\/v2\/posts\/1195","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/sisu.ut.ee\/ribolab\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/sisu.ut.ee\/ribolab\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/sisu.ut.ee\/ribolab\/wp-json\/wp\/v2\/users\/194"}],"replies":[{"embeddable":true,"href":"https:\/\/sisu.ut.ee\/ribolab\/wp-json\/wp\/v2\/comments?post=1195"}],"version-history":[{"count":3,"href":"https:\/\/sisu.ut.ee\/ribolab\/wp-json\/wp\/v2\/posts\/1195\/revisions"}],"predecessor-version":[{"id":1251,"href":"https:\/\/sisu.ut.ee\/ribolab\/wp-json\/wp\/v2\/posts\/1195\/revisions\/1251"}],"wp:attachment":[{"href":"https:\/\/sisu.ut.ee\/ribolab\/wp-json\/wp\/v2\/media?parent=1195"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/sisu.ut.ee\/ribolab\/wp-json\/wp\/v2\/categories?post=1195"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/sisu.ut.ee\/ribolab\/wp-json\/wp\/v2\/tags?post=1195"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}