Hfq-Antisense RNA I Binding Regulates RNase E-Dependent RNA Stability and ColE1 Plasmid Copy Number
Abstract
:1. Introduction
2. Results
2.1. Deletion of the RNA I 5′ End Single-Stranded Region Significantly Reduces Hfq–RNA I Binding, and High-Affinity Hfq Binding Limits RNase E Cleavage
2.2. RNA I Displays a Sub-Micromolar Binding Affinity for Hfq Protein In Vitro
2.3. Proximal Face Mutants of Hfq Decrease Both DsrA and RNA I Binding, and These Two RNAs Compete with Each Other for Hfq Binding
2.4. Hfq protects RNA I from RNase E-Mediated Degradation Predominantly through Its Proximal but Not Distal Face in E. coli
2.5. Hfq Controls ColE1 Plasmid Copy Number by Regulating RNA I Stability
3. Discussion
3.1. Hfq Might Have Other Binding Sites in Addition to the 5′ end Single-Stranded AU-Rich Region of RNA I
3.2. Hfq Binding Face for RNA I
3.3. How Mutations of Hfq Binding Surfaces Affect RNA I Stability
3.4. Possible Competition between Plasmid-Borne Antisense RNA and Chromosome-Borne sRNAs
4. Materials and Methods
4.1. Bacterial Strains and Plasmid Construction
4.2. Protein Expression and Purification
4.3. RNA Preparation, N-RNE In Vitro Cleavage, In Vitro Protein–RNA Gel-Shift Assay, and RNA–Protein Competition Assay
4.4. RNA Half-Life Determination, Total RNA Extraction, and Northern Blot Analysis
4.5. Plasmid Copy Number Determination
4.6. DNA Isolation and Quantitative PCR (qPCR)
4.7. Quantification and Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Wildtype | Proximal Face Mutants | Distal Face Mutant | ||||
---|---|---|---|---|---|---|
HfqWT | Q8A | K56A | H57A | K56/H57A | Y25D* | |
DsrA | 100% | 46.9 ± 4.0% b | 24.4 ± 4.3% a | 66.5 ± 17.0% d | 13.2 ± 6.9% b | 162.7 ± 13.7% c |
RNA I | 100% | 86.5 ± 3.6% b | 36.1 ± 5.1% b | 72.7 ± 5.0% c | 13.8 ± 8.6% b | 125.2 ± 22.4% d |
Primer Name | Primer Sequence | Purpose |
---|---|---|
Primer 1 | 5′-CCG CTC GAG AAT GGC TAA GGG GCA AT-3′ | pBAD-His-Hfq construction |
Primer 2 | 5′-CCG GAA TTC TTA TTC GGT TTC TTC GCT GTC CTG TTG-3′ | pBAD-His-Hfq construction |
Primer 3 | 5′-GGA ATT CCA TAT GGC TAA GGG GCA ATC TTT ACA-3′ | pBAD-Hfq construction |
Primer 4 | 5′-GGG CAA TCT TTA GCA GAT CCG TTC CTG AAC GCA CTG CG-3′ | Hfq Q8A mutagenesis |
Primer 5 | 5′-GTT CAG GAA CGG ATC TGC TAA AGA TTG CCC CTT AG-3′ | Hfq Q8A mutagenesis |
Primer 6 | 5′-GTT TCT ATT GAT TTG GTG AAT GGT ATT AAG CTG CAA GGG C-3′ | Hfq Y25D* mutagenesis |
Primer 7 | 5′-CCA TTC ACC AAA TCA ATA GAA ACT GGA ACA CG-3′ | Hfq Y25D* mutagenesis |
Primer 8 | 5′-GGT TTA CGC GCA CGC GAT TTC TAC TGT TGT CC-3′ | Hfq K56A mutagenesis |
Primer 9 | 5′-GAA ATC GCG TGC GCG TAA ACC ATC TGG CTG AC-3′ | Hfq K56A mutagenesis |
Primer 10 | 5′-CAG CCA GAT GGT TTA CAA GGC CGC GAT TTC TAC TGT TGT CC-3′ | Hfq H57A mutagenesis |
Primer 11 | 5′-GGA CAA CAG TAG AAA TCG CGG CCT TGT AAA CCA TCT GGC TG-3′ | Hfq H57A mutagenesis |
Primer 12 | 5′-GGT TTA CGC GGC CGC GAT TTC TAC TGT TGT CC-3′ | Hfq K56A/H57A mutagenesis |
Primer 13 | 5′-GAA ATC GCG GCC GCG TAA ACC ATC TGG CTG AC-3′ | Hfq K56A/H57A mutagenesis |
Primer 14 | 5′-TAA TAC GAC TCA CTA TAG GGA CAG TAT TTG GTA TCT GCG C-3′ | RNA IFL T7 template |
Primer 15 | 5′-ACA AAA AAA CCA CCG CTA CC-3′ | RNA IFL T7 template |
Primer 16 | 5′-TAA TAC GAC TCA CTA TAG GGT GGT ATC TGC GCT CTG C-3′ | RNA I-8 T7 template |
Primer 17 | 5′-TAA TAC GAC TCA CTA TAG GGA ACA CAT CAG ATT TCC TGG-3′ | DsrA T7 template |
Primer 18 | 5′-AAA TCC CGA CCC TGA GG-3′ | DsrA T7 template |
Primer 19 | 5′-GTA ACT GGC TTC AGC AGA GCG CAG ATA CC-3′ | RNA I oligo probe |
Primer 20 | CGG TTC TAT GGT GGT TGA | qPCR primer for mreB |
Primer 21 | GCG CAC AGA AGA GGA GTA | qPCR primer for mreB |
Primer 22 | TTC CGG CTG GCT GGT TTA TT | qPCR primer for ampA |
Primer 23 | TGA CTC CCC GTC GTG TAG AT | qPCR primer for ampA |
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Wang, W.-S.; Lin-Chao, S. Hfq-Antisense RNA I Binding Regulates RNase E-Dependent RNA Stability and ColE1 Plasmid Copy Number. Int. J. Mol. Sci. 2024, 25, 3955. https://doi.org/10.3390/ijms25073955
Wang W-S, Lin-Chao S. Hfq-Antisense RNA I Binding Regulates RNase E-Dependent RNA Stability and ColE1 Plasmid Copy Number. International Journal of Molecular Sciences. 2024; 25(7):3955. https://doi.org/10.3390/ijms25073955
Chicago/Turabian StyleWang, Wei-Syuan, and Sue Lin-Chao. 2024. "Hfq-Antisense RNA I Binding Regulates RNase E-Dependent RNA Stability and ColE1 Plasmid Copy Number" International Journal of Molecular Sciences 25, no. 7: 3955. https://doi.org/10.3390/ijms25073955