Protein–RNA recognition

Guzman, Roberto N. De; Turner, Ryan B.; Summers, Michael F.

doi:10.1002/(sici)1097-0282(1998)48:2<181::aid-bip7>3.0.co;2-l

Cited by 58 publications

(40 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Labeling ms2-RNA with MS2-GFP fusion protein is a general framework that can be extended to other specific RNA-binding proteins. The use of other RNAbinding proteins, such as the spliceosomal U1A protein (32,33), fused to fluorescent proteins of various colors would allow us to simultaneously measure transcriptional dynamics from more than one genetic element within the same cell. Most importantly, this plasmid system can be used in prokaryotes to report in real time the activity of any promoter controlling the expression of the recognition RNA sequence for RNA-binding proteins fused to GFP.…”

Section: Resultsmentioning

confidence: 99%

Real-time RNA profiling within a single bacterium

Harlepp

Guet

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Characterizing the dynamics of specific RNA levels requires realtime RNA profiling in a single cell. We show that the combination of a synthetic modular genetic system with fluorescence correlation spectroscopy allows us to directly measure in real time the activity of any specific promoter in prokaryotes. Using a simple inducible gene expression system, we found that induced RNA levels within a single bacterium of Escherichia coli exhibited a pulsating profile in response to a steady input of inducer. The genetic deletion of an efflux pump system, a key determinant of antibiotic resistance, altered the pulsating transcriptional dynamics and caused overexpression of induced RNA. In contrast with population measurements, real-time RNA profiling permits identifying relationships between genotypes and transcriptional dynamics that are accessible only at the level of the single cell.fluorescence correlation spectroscopy ͉ multidrug efflux ͉ transcription ͉ cell cycle ͉ noise N early half of a century ago, the discovery of messenger RNA as ''an unstable intermediate'' established RNA dynamics as one of the key properties of molecular adaptation in nature (1). Given the transient character of RNA transcripts, it has proved technically challenging to monitor in real time the transcription activity of a promoter within an individual prokaryotic cell (2-4). There exist various powerful in vitro methods for measuring RNA levels, including Northern blots, RT-PCR, and microarrays. Expression levels of a specific RNA species, extracted from population measurements, generally come from cells that are in different cell cycle states and exhibit different behaviors due to the variations of their internal biochemical parameters (5). Because of these inherent differences among single cells within a population, the transient dynamics of transcriptional networks may only be correctly characterized by monitoring transcriptional activity as a function of time within a single cell (6). Therefore, we need simple, noninvasive, real-time approaches to study the relationship between structure and dynamics of intracellular transcriptional networks in a single living cell. To this end, we constructed an in vivo synthetic genetic system that allows us to monitor the dynamics of a specific RNA species as a function of time within a single bacterium. Materials and MethodsFluorescence Correlation Spectroscopy (FCS) Apparatus. The incident excitation from a blue laser beam (Sapphire 488 nm, 20 mW, Coherent, Santa Clara, CA) is focused with a ϫ100 microscope objective lens (numerical aperture ϭ 1.3, Olympus, Melville, NY) onto a diffraction-limited spot in the bacterium. The emitted green fluorescence is collected in a confocal geometry and detected with an avalanche photo-diode (spcmaqr-16fc, PerkinElmer). The fluorescent signal is analyzed in real time with a fast correlator (5000EPP, ALV, Langen, Germany). We visualized the bacterium attached onto a glass coverslip by using a dark-field illumination. The temporal variations from the emitted lig...

show abstract

Section: Resultsmentioning

confidence: 99%

Real-time RNA profiling within a single bacterium

Harlepp

Guet

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…76 The protein was then extensively desalted using Centricon YM-3 ultrafiltration devices (Millipore, Billerica, MA) against ammonium acetate buffer of the desired concentration with pH adjusted to 7.0. The purity and integrity of the protein, including the presence of the coordinated zinc ions, were confirmed by ESI-FTMS.…”

Section: Nc and Rna Constructsmentioning

confidence: 99%

Dissecting the Protein–RNA and RNA–RNA Interactions in the Nucleocapsid-mediated Dimerization and Isomerization of HIV-1 Stemloop 1

Hagan¹,

Fabris²

2007

Journal of Molecular Biology

View full text Add to dashboard Cite

The specific binding of HIV-1 nucleocapsid protein (NC) to the different forms assumed in vitro by the stemloop 1 (Lai variant) of the genome's packaging signal has been investigated using electrospray ionization-Fourier transform mass spectrometry (ESI-FTMS). The simultaneous observation of protein-RNA and RNA-RNA interactions in solution has provided direct information about the role of NC in the two-step model of RNA dimerization and isomerization. In particular, two distinct binding sites have been identified on the monomeric stemloop structure, corresponding to the apical loop and stem-bulge motifs. These sites share similar binding affinities that are intermediate between those of stemloop 3 (SL3) and the putative stemloop 4 (SL4) of the packaging signal. Binding to the apical loop, which contains the dimerization initiation site (DIS), competes directly with the annealing of self-complementary sequences to form a metastable kissing-loop (KL) dimer. In contrast, binding to the stem-bulge affects indirectly the monomer-dimer equilibrium by promoting the rearrangement of KL into the more stable extended duplex (ED) conformer. This process is mediated by the duplex-melting activity of NC, which destabilizes the intramolecular base pairs surrounding the KL stem-bulges and enables their exchange to form the inter-strand pairs that define the ED structure. In this conformer, high-affinity binding takes place at stem-bulge sites that are identical to those present in the monomeric and KL forms. In this case, however, the NC-induced 'breathing' does not result in dissociation of the double-stranded structure because of the large number of intermolecular base pairs. The different binding modes manifested by conformer-specific mutants have shown that NC can also provide low affinity interactions with the bulged-out adenines flanking the DIS region of the ED conformer, thus supporting the hypothesis that these exposed nucleotides may constitute 'base-grips' for protein contacts during the late stages of the viral lifecycle.

show abstract

“…Interestingly, critical regions 1 and 2 were aligned in parallel and adjacent to each other within the three-dimensional space of the protein. In contrast to certain other multimeric RNA-binding proteins, such as the mammalian U1A and Nova (33,34), bacteriophage coat protein MS2 (35) and E. coli AspRS (36,37) wherein individual monomers comprise a complete functional domain, CsrA requires that each monomer contribute N-and C-terminal portions to the creation of a functional region. This further implies that a symmetrical CsrA homodimer contains two critical surfaces or subdomains located on opposite sides of the molecule.…”

mentioning

confidence: 99%

Comprehensive Alanine-scanning Mutagenesis ofEscherichia coliCsrA Defines Two Subdomains of Critical Functional Importance

Mercante¹,

Suzuki²,

Cheng³

et al. 2006

J. Biol. Chem.

105

181

View full text Add to dashboard Cite

The RNA-binding protein CsrA (carbon storage regulator) of Escherichia coli is a global regulator of gene expression and is representative of the CsrA/RsmA family of bacterial proteins. These proteins act by regulating mRNA translation and stability and are antagonized by binding to small noncoding RNAs. Although the RNA target sequence and structure for CsrA binding have been well defined, little information exists concerning the protein requirements for RNA recognition. The three-dimensional structures of three CsrA/RsmA proteins were recently solved, revealing a novel protein fold consisting of two interdigitated monomers. Here, we performed comprehensive alanine-scanning mutagenesis on csrA of E. coli and tested the 58 resulting mutants for regulation of glycogen accumulation, motility, and biofilm formation. Quantitative effects of these mutations on expression of glgCA-lacZ, flhDC-lacZ, and pgaA-lacZ translational fusions were also examined, and eight of the mutant proteins were purified and tested for RNA binding. These studies identified two regions of the amino acid sequence that were critical for regulation and RNA binding, located within the first (␤ 1 , residues 2-7) and containing the last (␤ 5 , residues 40 -47) ␤-strands of CsrA. The ␤ 1 and ␤ 5 strands of opposite monomers lie adjacent and parallel to each other in the three-dimensional structure of this protein. Given the symmetry of the CsrA dimer, these findings imply that two distinct RNA binding surfaces or functional subdomains lie on opposite sides of the protein.

show abstract

Protein–RNA recognition

Cited by 58 publications

References 50 publications

Real-time RNA profiling within a single bacterium

Real-time RNA profiling within a single bacterium

Dissecting the Protein–RNA and RNA–RNA Interactions in the Nucleocapsid-mediated Dimerization and Isomerization of HIV-1 Stemloop 1

Comprehensive Alanine-scanning Mutagenesis ofEscherichia coliCsrA Defines Two Subdomains of Critical Functional Importance

Contact Info

Product

Resources

About