Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli
The anti-Shine-Dalgarno (ASD) sequence of 16S rRNA is highly conserved across Bacteria, and yet usage of Shine-Dalgarno (SD) sequences in mRNA varies dramatically, depending on the lineage. Here, we compared the effects of ASD mutagenesis in Escherichia coli, a Gammaproteobacteria which commonly emp...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2021-12-01
|
| Series: | Frontiers in Molecular Biosciences |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmolb.2021.787388/full |
| _version_ | 1818459147110187008 |
|---|---|
| author | Zakkary A. McNutt Zakkary A. McNutt Mai D. Gandhi Elan A. Shatoff Elan A. Shatoff Bappaditya Roy Bappaditya Roy Aishwarya Devaraj Aishwarya Devaraj Ralf Bundschuh Ralf Bundschuh Ralf Bundschuh Ralf Bundschuh Kurt Fredrick Kurt Fredrick Kurt Fredrick |
| author_facet | Zakkary A. McNutt Zakkary A. McNutt Mai D. Gandhi Elan A. Shatoff Elan A. Shatoff Bappaditya Roy Bappaditya Roy Aishwarya Devaraj Aishwarya Devaraj Ralf Bundschuh Ralf Bundschuh Ralf Bundschuh Ralf Bundschuh Kurt Fredrick Kurt Fredrick Kurt Fredrick |
| author_sort | Zakkary A. McNutt |
| collection | DOAJ |
| description | The anti-Shine-Dalgarno (ASD) sequence of 16S rRNA is highly conserved across Bacteria, and yet usage of Shine-Dalgarno (SD) sequences in mRNA varies dramatically, depending on the lineage. Here, we compared the effects of ASD mutagenesis in Escherichia coli, a Gammaproteobacteria which commonly employs SD sequences, and Flavobacterium johnsoniae, a Bacteroidia which rarely does. In E. coli, 30S subunits carrying any single substitution at positions 1,535–1,539 confer dominant negative phenotypes, whereas subunits with mutations at positions 1,540–1,542 are sufficient to support cell growth. These data suggest that CCUCC (1,535–1,539) represents the functional core of the element in E. coli. In F. johnsoniae, deletion of three ribosomal RNA (rrn) operons slowed growth substantially, a phenotype largely rescued by a plasmid-borne copy of the rrn operon. Using this complementation system, we found that subunits with single mutations at positions 1,535–1,537 are as active as control subunits, in sharp contrast to the E. coli results. Moreover, subunits with quadruple substitution or complete replacement of the ASD retain substantial, albeit reduced, activity. Sedimentation analysis revealed that these mutant subunits are overrepresented in the subunit fractions and underrepresented in polysome fractions, suggesting some defect in 30S biogenesis and/or translation initiation. Nonetheless, our collective data indicate that the ASD plays a much smaller role in F. johnsoniae than in E. coli, consistent with SD usage in the two organisms. |
| first_indexed | 2024-12-14T23:09:44Z |
| format | Article |
| id | doaj.art-efb63cdf994e44a9b2aaa0018c37b2a3 |
| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-12-14T23:09:44Z |
| publishDate | 2021-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-efb63cdf994e44a9b2aaa0018c37b2a32022-12-21T22:44:13ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2021-12-01810.3389/fmolb.2021.787388787388Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coliZakkary A. McNutt0Zakkary A. McNutt1Mai D. Gandhi2Elan A. Shatoff3Elan A. Shatoff4Bappaditya Roy5Bappaditya Roy6Aishwarya Devaraj7Aishwarya Devaraj8Ralf Bundschuh9Ralf Bundschuh10Ralf Bundschuh11Ralf Bundschuh12Kurt Fredrick13Kurt Fredrick14Kurt Fredrick15Ohio State Biochemistry Program, The Ohio State University, Columbus, OH, United StatesCenter for RNA Biology, The Ohio State University, Columbus, OH, United StatesDepartment of Microbiology, The Ohio State University, Columbus, OH, United StatesCenter for RNA Biology, The Ohio State University, Columbus, OH, United StatesDepartment of Physics, The Ohio State University, Columbus, OH, United StatesCenter for RNA Biology, The Ohio State University, Columbus, OH, United StatesDepartment of Microbiology, The Ohio State University, Columbus, OH, United StatesOhio State Biochemistry Program, The Ohio State University, Columbus, OH, United StatesCenter for RNA Biology, The Ohio State University, Columbus, OH, United StatesCenter for RNA Biology, The Ohio State University, Columbus, OH, United StatesDepartment of Physics, The Ohio State University, Columbus, OH, United StatesDepartment of Chemistry and Biochemistry, The Ohio State University, Columbus, OH, United, StatesDivision of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, OH, United StatesOhio State Biochemistry Program, The Ohio State University, Columbus, OH, United StatesCenter for RNA Biology, The Ohio State University, Columbus, OH, United StatesDepartment of Microbiology, The Ohio State University, Columbus, OH, United StatesThe anti-Shine-Dalgarno (ASD) sequence of 16S rRNA is highly conserved across Bacteria, and yet usage of Shine-Dalgarno (SD) sequences in mRNA varies dramatically, depending on the lineage. Here, we compared the effects of ASD mutagenesis in Escherichia coli, a Gammaproteobacteria which commonly employs SD sequences, and Flavobacterium johnsoniae, a Bacteroidia which rarely does. In E. coli, 30S subunits carrying any single substitution at positions 1,535–1,539 confer dominant negative phenotypes, whereas subunits with mutations at positions 1,540–1,542 are sufficient to support cell growth. These data suggest that CCUCC (1,535–1,539) represents the functional core of the element in E. coli. In F. johnsoniae, deletion of three ribosomal RNA (rrn) operons slowed growth substantially, a phenotype largely rescued by a plasmid-borne copy of the rrn operon. Using this complementation system, we found that subunits with single mutations at positions 1,535–1,537 are as active as control subunits, in sharp contrast to the E. coli results. Moreover, subunits with quadruple substitution or complete replacement of the ASD retain substantial, albeit reduced, activity. Sedimentation analysis revealed that these mutant subunits are overrepresented in the subunit fractions and underrepresented in polysome fractions, suggesting some defect in 30S biogenesis and/or translation initiation. Nonetheless, our collective data indicate that the ASD plays a much smaller role in F. johnsoniae than in E. coli, consistent with SD usage in the two organisms.https://www.frontiersin.org/articles/10.3389/fmolb.2021.787388/fullribosometranslationRF2 (prfB)initiationbacteroidetes |
| spellingShingle | Zakkary A. McNutt Zakkary A. McNutt Mai D. Gandhi Elan A. Shatoff Elan A. Shatoff Bappaditya Roy Bappaditya Roy Aishwarya Devaraj Aishwarya Devaraj Ralf Bundschuh Ralf Bundschuh Ralf Bundschuh Ralf Bundschuh Kurt Fredrick Kurt Fredrick Kurt Fredrick Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli Frontiers in Molecular Biosciences ribosome translation RF2 (prfB) initiation bacteroidetes |
| title | Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli |
| title_full | Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli |
| title_fullStr | Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli |
| title_full_unstemmed | Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli |
| title_short | Comparative Analysis of anti-Shine- Dalgarno Function in Flavobacterium johnsoniae and Escherichia coli |
| title_sort | comparative analysis of anti shine dalgarno function in flavobacterium johnsoniae and escherichia coli |
| topic | ribosome translation RF2 (prfB) initiation bacteroidetes |
| url | https://www.frontiersin.org/articles/10.3389/fmolb.2021.787388/full |
| work_keys_str_mv | AT zakkaryamcnutt comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT zakkaryamcnutt comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT maidgandhi comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT elanashatoff comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT elanashatoff comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT bappadityaroy comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT bappadityaroy comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT aishwaryadevaraj comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT aishwaryadevaraj comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT ralfbundschuh comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT ralfbundschuh comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT ralfbundschuh comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT ralfbundschuh comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT kurtfredrick comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT kurtfredrick comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli AT kurtfredrick comparativeanalysisofantishinedalgarnofunctioninflavobacteriumjohnsoniaeandescherichiacoli |