Bulged-out nucleotides in an antisense RNA are required for rapid target RNA binding in vitro and inhibition in vivo
(1995) In Nucleic Acids Research 23(4). p.580-587- Abstract
Naturally occurring antisense RNAs in prokaryotes are generally short, highly structured and untranslated. Stem-loops are always present, and loop regions serve as primary recognition structures in most cases. Single-stranded tails or internal unstructured regions are required for initiation of stable pairing between antisense and target RNA. Most antisense RNAs contain bulged-out nucleotides or small internal loops in upper stem regions. Here we investigated the role of the bulged-out nucleotides of CopA (the copy number regulator of plasmid R1) in determining the binding properties of this antisense RNA to its target in vitro and the efficiency of a translational inhibition in vivo. The introduction of perfect helicity in the region... (More)
Naturally occurring antisense RNAs in prokaryotes are generally short, highly structured and untranslated. Stem-loops are always present, and loop regions serve as primary recognition structures in most cases. Single-stranded tails or internal unstructured regions are required for initiation of stable pairing between antisense and target RNA. Most antisense RNAs contain bulged-out nucleotides or small internal loops in upper stem regions. Here we investigated the role of the bulged-out nucleotides of CopA (the copy number regulator of plasmid R1) in determining the binding properties of this antisense RNA to its target in vitro and the efficiency of a translational inhibition in vivo. The introduction of perfect helicity in the region of the two bulges in CopA decreased pairing rate constants by up to 180-fold, increased equilibrium dissociation constants of the 'kissing intermediate' up to 14-fold, and severely impaired inhibition of repA expression. A previously described loop size mutant of CopA showed decreased pairing rates, but, in contrast to the bulge-less mutant CopAs, shows a decreased dissociation constant of the 'kissing complex'. We conclude that removal of the specific bulges/internal loops within the stem-loop II of CopA impairs the inhibitor, and that creation of an internal loop at a different position does not restore activity, emphasizing the optimal folding of wild-type CopA. The accompanying paper shows that an additional function of bulges can be protection from RNase III cleavage.
(Less)
- author
- Hjalt, Tord Å. H. LU and Wagner, E.G.H.
- publishing date
- 1995-02-25
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Bacterial Proteins/biosynthesis, Bacteriocin Plasmids/genetics, Binding Sites, DNA Helicases, DNA-Binding Proteins, Escherichia coli/genetics, Kinetics, Macromolecular Substances, Nucleic Acid Conformation, Proteins, R Factors/genetics, RNA, Antisense/chemistry, RNA, Bacterial/chemistry, Recombinant Fusion Proteins/biosynthesis, Trans-Activators
- in
- Nucleic Acids Research
- volume
- 23
- issue
- 4
- pages
- 8 pages
- publisher
- Oxford University Press
- external identifiers
-
- scopus:0028950890
- pmid:7534907
- ISSN
- 0305-1048
- DOI
- 10.1093/nar/23.4.580
- language
- English
- LU publication?
- no
- id
- d6fce06d-a5b5-49bd-8930-9df893a11a77
- date added to LUP
- 2023-11-16 11:36:38
- date last changed
- 2024-01-14 04:13:12
@article{d6fce06d-a5b5-49bd-8930-9df893a11a77,
abstract = {{<p>Naturally occurring antisense RNAs in prokaryotes are generally short, highly structured and untranslated. Stem-loops are always present, and loop regions serve as primary recognition structures in most cases. Single-stranded tails or internal unstructured regions are required for initiation of stable pairing between antisense and target RNA. Most antisense RNAs contain bulged-out nucleotides or small internal loops in upper stem regions. Here we investigated the role of the bulged-out nucleotides of CopA (the copy number regulator of plasmid R1) in determining the binding properties of this antisense RNA to its target in vitro and the efficiency of a translational inhibition in vivo. The introduction of perfect helicity in the region of the two bulges in CopA decreased pairing rate constants by up to 180-fold, increased equilibrium dissociation constants of the 'kissing intermediate' up to 14-fold, and severely impaired inhibition of repA expression. A previously described loop size mutant of CopA showed decreased pairing rates, but, in contrast to the bulge-less mutant CopAs, shows a decreased dissociation constant of the 'kissing complex'. We conclude that removal of the specific bulges/internal loops within the stem-loop II of CopA impairs the inhibitor, and that creation of an internal loop at a different position does not restore activity, emphasizing the optimal folding of wild-type CopA. The accompanying paper shows that an additional function of bulges can be protection from RNase III cleavage.</p>}},
author = {{Hjalt, Tord Å. H. and Wagner, E.G.H.}},
issn = {{0305-1048}},
keywords = {{Bacterial Proteins/biosynthesis; Bacteriocin Plasmids/genetics; Binding Sites; DNA Helicases; DNA-Binding Proteins; Escherichia coli/genetics; Kinetics; Macromolecular Substances; Nucleic Acid Conformation; Proteins; R Factors/genetics; RNA, Antisense/chemistry; RNA, Bacterial/chemistry; Recombinant Fusion Proteins/biosynthesis; Trans-Activators}},
language = {{eng}},
month = {{02}},
number = {{4}},
pages = {{580--587}},
publisher = {{Oxford University Press}},
series = {{Nucleic Acids Research}},
title = {{Bulged-out nucleotides in an antisense RNA are required for rapid target RNA binding in vitro and inhibition in vivo}},
url = {{http://dx.doi.org/10.1093/nar/23.4.580}},
doi = {{10.1093/nar/23.4.580}},
volume = {{23}},
year = {{1995}},
}