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Lee Gehrke - One of the best experts on this subject based on the ideXlab platform.
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Coat Protein Activation of Alfalfa Mosaic Virus Replication Is Concentration Dependent
Journal of virology, 2005Co-Authors: Laura M. Guogas, Siana M. Laforest, Lee GehrkeAbstract:Alfalfa mosaic virus (AMV) and ilarvirus RNAs are infectious only in the presence of the viral Coat Protein; therefore, an understanding of Coat Protein's function is important for defining viral replication mechanisms. Based on in vitro replication experiments, the conformational switch model states that AMV Coat Protein blocks minus-strand RNA synthesis (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999), while another report states that Coat Protein present in an inoculum is required to permit minus-strand synthesis (L. Neeleman and J. F. Bol, Virology 254:324-333, 1999). Here, we report on experiments that address these contrasting results with a goal of defining Coat Protein's function in the earliest stages of AMV replication. To detect Coat-Protein-activated AMV RNA replication, we designed and characterized a subgenomic luciferase reporter construct. We demonstrate that activation of viral RNA replication by Coat Protein is concentration dependent; that is, replication was strongly stimulated at low Coat Protein concentrations but decreased progressively at higher concentrations. Genomic RNA3 mutations preventing Coat Protein mRNA translation or disrupting Coat Protein's RNA binding domain diminished replication. The data indicate that RNA binding and an ongoing supply of Coat Protein are required to initiate replication on progeny genomic RNA transcripts. The data do not support the conformational switch model's claim that Coat Protein inhibits the initial stages of viral RNA replication. Replication activation may correlate with low local Coat Protein concentrations and low Coat Protein occupancy on the multiple binding sites present in the 3' untranslated regions of the viral RNAs.
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viral Coat Protein peptides with limited sequence homology bind similar domains of alfalfa mosaic virus and tobacco streak virus rnas
Journal of Virology, 1998Co-Authors: M M Swanson, Lee Gehrke, Patricia Anselmckinney, Felicia Houserscott, Vidadi Yusibov, Sue L LoeschfriesAbstract:An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral Coat Protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV Coat Protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV Coat Protein will activate AMV RNA replication and vice versa. Although AMV and TSV Coat Proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077-5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3'-terminal TSV and AMV RNA fragments. The arginine-rich TSV Coat Protein peptide binds viral RNA with lower affinity than the lysine-rich AMV Coat Protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3'-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind Coat Protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus Coat Proteins.
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Nucleotide sequence and structural determinants of specific binding of Coat Protein or Coat Protein peptides to the 3' untranslated region of alfalfa mosaic virus RNA 4.
Journal of virology, 1994Co-Authors: F Houser-scott, M L Baer, K F Liem, J M Cai, Lee GehrkeAbstract:The specific binding of alfalfa mosaic virus Coat Protein to viral RNA requires determinants in the 3' untranslated region (UTR). Coat Protein and peptide binding sites in the 3' UTR of alfalfa mosaic virus RNA 4 have been analyzed by hydroxyl radical footprinting, deletion mapping, and site-directed mutagenesis experiments. The 3' UTR has several stable hairpins that are flanked by single-stranded (A/U)UGC sequences. Hydroxyl radical footprinting data show that five sites in the 3' UTR of alfalfa mosaic virus RNA 4 are protected by Coat Protein, and four of the five protected regions contain AUGC or UUGC. Electrophoretic mobility band shift results suggest four Coat Protein binding sites in the 3' UTR. A 3'-terminal 39-nucleotide RNA fragment containing four AUGC repeats bound Coat Protein and Coat Protein peptides with high affinity; however, Coat Protein bound poorly to antisense 3' UTR transcripts and poly(AUGC)10. Site-directed mutagenesis of AUGC865-868 resulted in a loss of Coat Protein binding and peptide binding by the RNA fragment. Alignment of alfalfa mosaic RNA sequences with those from several closely related ilarviruses demonstrates that AUGC865-868 is perfectly conserved; moreover, the RNAs are predicted to form similar 3'-terminal secondary structures. The data strongly suggest that alfalfa mosaic virus Coat Protein and ilavirus Coat Proteins recognize invariant AUGC sequences in the context of conserved structural elements.
Carolyn M Teschke - One of the best experts on this subject based on the ideXlab platform.
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conformational changes in bacteriophage p22 scaffolding Protein induced by interaction with Coat Protein
Journal of Molecular Biology, 2011Co-Authors: Pauline G Padillameier, Carolyn M TeschkeAbstract:Abstract Many prokaryotic and eukaryotic double-stranded DNA viruses use a scaffolding Protein to assemble their capsid. Assembly of the double-stranded DNA bacteriophage P22 procapsids requires the interaction of 415 molecules of Coat Protein and 60–300 molecules of scaffolding Protein. Although the 303-amino-acid scaffolding Protein is essential for proper assembly of procapsids, little is known about its structure beyond an NMR structure of the extreme C-terminus, which is known to interact with Coat Protein. Deletion mutagenesis indicates that other regions of scaffolding Protein are involved in interactions with Coat Protein and other capsid Proteins. Single-cysteine and double-cysteine variants of scaffolding Protein were generated for use in fluorescence resonance energy transfer and cross-linking experiments designed to probe the conformation of scaffolding Protein in solution and within procapsids. We showed that the N-terminus and the C-terminus are proximate in solution, and that the middle of the Protein is near the N-terminus but not accessible to the C-terminus. In procapsids, the N-terminus was no longer accessible to the C-terminus, indicating that there is a conformational change in scaffolding Protein upon assembly. In addition, our data are consistent with a model where scaffolding Protein dimers are positioned parallel with one another with the associated C-termini.
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determinants of bacteriophage p22 polyhead formation the role of Coat Protein flexibility in conformational switching
Molecular Microbiology, 2010Co-Authors: Margaret M Suhanovsky, Kristin N Parent, Sarah E Dunn, Timothy S Baker, Carolyn M TeschkeAbstract:Summary We have investigated determinants of polyhead for- mation in bacteriophage P22 in order to understand the molecular mechanism by which Coat Protein assembly goes astray. Polyhead assembly is caused by amino acid substitutions in Coat Protein at position 170, which is located in the b-hinge. In vivo scaffold- ing Protein does not correct polyhead assembly by F170A or F170K Coat Proteins, but does for F170L. All F170 variants bind scaffolding Protein more weakly than wild-type as observed by affinity chromatogra- phy with scaffolding Protein-agarose and scaffolding Protein shell re-entry experiments. Electron cryo- microscopy and three-dimensional image reconstruc- tions of F170A and F170K empty procapsid shells showed that there is a decreased flexibility of the Coat subunits relative to wild-type. This was confirmed by limited proteolysis and Protein sequencing, which showed increased protection of the A-domain. Our data support the conclusion that the decrease in flex- ibility of the A-domain leads to crowding of the sub- units at the centre of the pentons, thereby favouring the hexon configuration during assembly. Thus, correct Coat Protein interactions with scaffolding Protein and maintenance of sufficient Coat Protein flexibility are crucial for proper P22 assembly. The Coat Protein b-hinge region is the major determinant for both features.
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phage p22 procapsids equilibrate with free Coat Protein subunits
Journal of Molecular Biology, 2007Co-Authors: Kristin N Parent, Margaret M Suhanovsky, Carolyn M TeschkeAbstract:Assembly of bacteriophage P22 procapsids has long served as a model for assembly of spherical viruses. Historically, assembly of viruses has been treated as a non-equilibrium process. Recently alternative models have been developed that treat spherical virus assembly as an equilibrium process. Here we have investigated whether P22 procapsids assembly reactions achieve equilibrium or are irreversibly trapped. To assemble a procapsid-like particle in vitro, pure Coat Protein monomers are mixed with scaffolding Protein. Here we present data that show that free subunits can exchange with assembled structures, indicating that assembly is a reversible, equilibrium process. When empty procapsid shells (procapsids with the scaffolding Protein stripped out) were diluted so that the concentration was below the dissociation constant (~5 μM) for Coat Protein monomers, free monomers were detected. The released monomers were assembly-competent; when NaCl was added to metastable partial capsids that were aged for an extended period, the Coat subunits were able to rapidly re-distribute from the partial capsids and form whole procapsids. Lastly, radioactive monomeric Coat subunits were able to exchange with the subunits from empty procapsid shells. The data presented here illustrate that Coat Protein monomers are able to dissociate from procapsids in an active state, that assembly of procapsids is consistent with reactions at equilibrium and follows the law of mass action.
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folding of phage p22 Coat Protein monomers kinetic and thermodynamic properties
Virology, 2003Co-Authors: Eric Anderson, Carolyn M TeschkeAbstract:To assemble into a virus with icosahedral symmetry, capsid Proteins must be able to attain multiple conformations. Whether this conformational diversity is achieved during folding of the subunit, or subsequently during assembly, is not clear. Phage P22 Coat Protein offers an ideal model to investigate the folding of a monomeric capsid subunit since its folding is independent of assembly. Our early studies indicated that P22 Coat Protein monomers could be folded into an assembly-competent state in vitro, with evidence of a kinetic intermediate. Using urea denaturation, Coat Protein monomers are shown to be marginally stable. The reversible folding of Coat Protein follows a three-state model, N ⇔ I ⇔ U, with an intermediate exhibiting most of the tryptophan fluorescence of the folded state, but little secondary structure. Folding and unfolding kinetics monitored by circular dichroism, tryptophan fluorescence, and bisANS fluorescence indicate that several kinetic intermediates are populated sequentially through parallel channels en route to the native state. Additionally, two native states were identified, suggesting that the several conformers required to assemble an icosahedral capsid may be found in solution before assembly ensues.
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single amino acid substitutions globally suppress the folding defects of temperature sensitive folding mutants of phage p22 Coat Protein
Journal of Biological Chemistry, 1999Co-Authors: Lili A Aramli, Carolyn M TeschkeAbstract:Abstract The amino acid sequence of a polypeptide defines both the folding pathway and the final three-dimensional structure of a Protein. Eighteen amino acid substitutions have been identified in bacteriophage P22 Coat Protein that are defective in folding and cause their folding intermediates to be substrates for GroEL and GroES. These temperature-sensitive folding (tsf) substitutions identify amino acids that are critical for directing the folding of Coat Protein. Additional amino acid residues that are critical to the folding process of P22 Coat Protein were identified by isolating second site suppressors of the tsf Coat Proteins. Suppressor substitutions isolated from the phage carrying the tsf Coat Protein substitutions included global suppressors, which are substitutions capable of alleviating the folding defects of numerous tsf Coat Protein mutants. In addition, potential global and site-specific suppressors were isolated, as well as a group of same site amino acid substitutions that had a less severe phenotype than the tsf parent. The global suppressors were located at positions 163, 166, and 170 in the Coat Protein sequence and were 8–190 amino acid residues away from the tsf parent. Although the folding of Coat Proteins with tsf amino acid substitutions was improved by the global suppressor substitutions, GroEL remained necessary for folding. Therefore, we believe that the global suppressor sites identify a region that is critical to the folding of Coat Protein.
Margaret M Suhanovsky - One of the best experts on this subject based on the ideXlab platform.
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multiple functional roles of the accessory i domain of bacteriophage p22 Coat Protein revealed by nmr structure and cryoem modeling
Structure, 2014Co-Authors: Alessandro A Rizzo, Margaret M Suhanovsky, Latasha C R Fraser, Matthew L Baker, Lisa M Jones, Don L Rempel, Michael L Gross, Wah Chiu, Andrei T AlexandrescuAbstract:Summary Some capsid Proteins built on the ubiquitous HK97-fold have accessory domains imparting specific functions. Bacteriophage P22 Coat Protein has a unique insertion domain (I-domain). Two prior I-domain models from subnanometer cryoelectron microscopy (cryoEM) reconstructions differed substantially. Therefore, the I-domain's nuclear magnetic resonance structure was determined and also used to improve cryoEM models of Coat Protein. The I-domain has an antiparallel six-stranded β-barrel fold, not previously observed in HK97-fold accessory domains. The D-loop, which is dynamic in the isolated I-domain and intact monomeric Coat Protein, forms stabilizing salt bridges between adjacent capsomers in procapsids. The S-loop is important for capsid size determination, likely through intrasubunit interactions. Ten of 18 Coat Protein temperature-sensitive-folding substitutions are in the I-domain, indicating its importance in folding and stability. Several are found on a positively charged face of the β-barrel that anchors the I-domain to a negatively charged surface of the Coat Protein HK97-core.
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an intramolecular chaperone inserted in bacteriophage p22 Coat Protein mediates its chaperonin independent folding
Journal of Biological Chemistry, 2013Co-Authors: Margaret M SuhanovskyAbstract:The bacteriophage P22 Coat Protein has the common HK97-like fold but with a genetically inserted domain (I-domain). The role of the I-domain, positioned at the outermost surface of the capsid, is unknown. We hypothesize that the I-domain may act as an intramolecular chaperone because the Coat Protein folds independently, and many folding mutants are localized to the I-domain. The function of the I-domain was investigated by generating the Coat Protein core without its I-domain and the isolated I-domain. The core Coat Protein shows a pronounced folding defect. The isolated I-domain folds autonomously and has a high thermodynamic stability and fast folding kinetics in the presence of a peptidyl prolyl isomerase. Thus, the I-domain provides thermodynamic stability to the full-length Coat Protein so that it can fold reasonably efficiently while still allowing the HK97-like core to retain the flexibility required for conformational switching during procapsid assembly and maturation.
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nmr assignments for the telokin like domain of bacteriophage p22 Coat Protein
Biomolecular Nmr Assignments, 2013Co-Authors: Alessandro A Rizzo, Margaret M Suhanovsky, Latasha C R Fraser, Sarah R Sheftic, Andrei T AlexandrescuAbstract:The bacteriophage P22 virion is assembled from identical Coat Protein monomers in a complex reaction that is generally conserved among tailed, double-stranded DNA bacteriophages and viruses. Many Coat Proteins of dsDNA viruses have structures based on the HK97 fold, but in some viruses and phages there are additional domains. In the P22 Coat Protein, a “telokin-like” domain was recently identified, whose structure has not yet been characterized at high-resolution. Two recently published low-resolution cryo-EM reconstructions suggest markedly different folds for the telokin-like domain that lead to alternative conclusions about its function in capsid assembly and stability. Here we report 1H, 15N, and 13C NMR resonance assignments for the telokin-like domain. The secondary structure predicted from the chemical shift values obtained in this work shows significant discrepancies from both cryo-EM models but agrees better with one of the models. In particular, the functionally important “D-loop” in one model shows chemical shifts and solvent exchange protection more consistent with β-sheet structure. Our work will set the basis for a high-resolution NMR structure determination of the telokin-like domain that will help improve the cryo-EM models, and in turn lead to a better understanding of how Coat Protein monomers assemble into the icosahedral capsids required for virulence.
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bacteriophage p22 capsid size determination roles for the Coat Protein telokin like domain and the scaffolding Protein amino terminus
Virology, 2011Co-Authors: Margaret M SuhanovskyAbstract:Assembly of icosahedral capsids of proper size and symmetry is not understood. Residue F170 in bacteriophage P22 Coat Protein is critical for conformational switching during assembly. Substitutions at this site cause assembly of tubes of hexamerically arranged Coat Protein. Intragenic suppressors of the ts phenotype of F170A and F170K Coat Protein mutants were isolated. Suppressors were repeatedly found in the Coat Protein telokin-like domain at position 285, which caused Coat Protein to assemble into petite procapsids and capsids. Petite capsid assembly strongly correlated to the side chain volume of the substituted amino acid. We hypothesize that larger side chains at position 285 torque the telokin-like domain, changing flexibility of the subunit and intercapsomer contacts. Thus, a single amino acid substitution in Coat Protein is sufficient to change capsid size. In addition, the products of assembly of the variant Coat Proteins were affected by the size of the internal scaffolding Protein.
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determinants of bacteriophage p22 polyhead formation the role of Coat Protein flexibility in conformational switching
Molecular Microbiology, 2010Co-Authors: Margaret M Suhanovsky, Kristin N Parent, Sarah E Dunn, Timothy S Baker, Carolyn M TeschkeAbstract:Summary We have investigated determinants of polyhead for- mation in bacteriophage P22 in order to understand the molecular mechanism by which Coat Protein assembly goes astray. Polyhead assembly is caused by amino acid substitutions in Coat Protein at position 170, which is located in the b-hinge. In vivo scaffold- ing Protein does not correct polyhead assembly by F170A or F170K Coat Proteins, but does for F170L. All F170 variants bind scaffolding Protein more weakly than wild-type as observed by affinity chromatogra- phy with scaffolding Protein-agarose and scaffolding Protein shell re-entry experiments. Electron cryo- microscopy and three-dimensional image reconstruc- tions of F170A and F170K empty procapsid shells showed that there is a decreased flexibility of the Coat subunits relative to wild-type. This was confirmed by limited proteolysis and Protein sequencing, which showed increased protection of the A-domain. Our data support the conclusion that the decrease in flex- ibility of the A-domain leads to crowding of the sub- units at the centre of the pentons, thereby favouring the hexon configuration during assembly. Thus, correct Coat Protein interactions with scaffolding Protein and maintenance of sufficient Coat Protein flexibility are crucial for proper P22 assembly. The Coat Protein b-hinge region is the major determinant for both features.
Peter E Prevelige - One of the best experts on this subject based on the ideXlab platform.
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the role of the Coat Protein a domain in p22 bacteriophage maturation
Viruses, 2014Co-Authors: David S Morris, Peter E PreveligeAbstract:Bacteriophage P22 has long been considered a hallmark model for virus assembly and maturation. Repurposing of P22 and other similar virus structures for nanotechnology and nanomedicine has reinvigorated the need to further understand the Protein-Protein interactions that allow for the assembly, as well as the conformational shifts required for maturation. In this work, gp5, the major Coat structural Protein of P22, has been manipulated in order to examine the mutational effects on procapsid stability and maturation. Insertions to the P22 Coat Protein A-domain, while widely permissive of procapsid assembly, destabilize the interactions necessary for virus maturation and potentially allow for the tunable adjustment of procapsid stability. Future manipulation of this region of the Coat Protein subunit can potentially be used to alter the stability of the capsid for controllable disassembly.
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identification and characterization of the domain structure of bacteriophage p22 Coat Protein
Biochemistry, 1999Co-Authors: Jason Lanman, Roman Tuma, Peter E PreveligeAbstract:The bacteriophage P22 serves as a model for assembly of icosahedral dsDNA viruses. The P22 procapsid, which constitutes the precursor for DNA packaging, is built from 420 copies of a single Coat Protein with the aid of stoichiometric amounts of scaffolding Protein. Upon DNA entry, the procapsid shell expands and matures into a stable virion. It was proposed that expansion is mediated by hinge bending and domain movement. We have used limited proteolysis to map the dynamic stability of the Coat Protein domain structures. The Coat Protein monomer is susceptible to proteolytic digestion, but limited proteolysis by small quantities of elastase or chymotrypsin yielded two metastable fragments (domains). The N-terminal domain (residues 1−180) is linked to the C-terminal domain (residues 205−429) by a protease-susceptible loop (residues 180−205). The two domains remain associated after the loop cleavage. Although only a small change of secondary structure results from the loop cleavage, both tertiary interdomain...
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electrostatic interactions drive scaffolding Coat Protein binding and procapsid maturation in bacteriophage p22
Virology, 1998Co-Authors: Matthew H Parker, Peter E PreveligeAbstract:The first step in assembly of the bacteriophage P22 is the formation of a T=7 icosahedral “procapsid,” the major components of which are the Coat Protein and an inner core composed of the scaffolding Protein. Although not present in the mature virion, the scaffolding Protein is required for procapsid assembly. Eleven amino-acid residues at the extreme carboxyl terminus of the scaffolding Protein are required for binding to the Coat Protein, and upon deletion of these residues, approximately 20 additional residues become disordered. Sequence analysis and NMR data suggest that the 30 residues at the carboxyl terminus form a helix-loop-helix motif which is stabilized by interhelical hydrophobic interactions. This “Coat Protein recognition domain” presents an unusually high number of positively charged residues on one face, suggesting that electrostatic interactions between this domain and the Coat Protein may contribute to recognition and binding. We report here that high ionic strength (1 M NaCl) completely inhibited procapsid assemblyin vitro.When scaffolding Protein was added to empty procapsid “shells” of Coat Protein, 1 M NaCl partially inhibited the binding of scaffolding Protein to the shells. This suggests that the positively charged Coat Protein recognition domain at the carboxyl terminus of the scaffolding Protein binds to a negatively charged region on the Coat Protein. During DNA packaging, the scaffolding Protein exits the procapsid; scaffolding Protein exit is followed by the expansion of the procapsid into a mature capsid. Procapsid shells can be induced to undergo a similar expansion reactionin vitroby heating (45–70°C); this process was also inhibited by 1 M NaCl. These results are consistent with a model in which negatively charged scaffold Protein-binding domains in the Coat Proteins move apart during procapsid expansion; this relief of electrostatic repulsion could provide a driving force for expansion and subsequent maturation. High-salt concentrations would screen this repulsion, while packaging of DNA (a polyanion)in vivomay increase the instability of the procapsid enough to trigger its expansion.
David S. Peabody - One of the best experts on this subject based on the ideXlab platform.
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Encapsidation of heterologous RNAs by bacteriophage MS2 Coat Protein
Nucleic acids research, 1993Co-Authors: Gavin Pickett, David S. PeabodyAbstract:The RNA bacteriophages of E. coli specifically encapsidate a single copy of the viral genome in a Protein shell composed mainly of 180 molecules of Coat Protein. Coat Protein is also a translational repressor and shuts off viral replicase synthesis by interaction with a RNA stem-loop containing the replicase initiation codon. We wondered whether the translational operator also serves as the viral pac site, the signal which mediates the exclusive encapsidation of viral RNA by its interaction with Coat Protein. To test this idea we measured the ability of lacZ RNA fused to the translational operator to be incorporated into virus-like particles formed from Coat Protein expressed from a plasmid. The results indicate that the operator-lacZ RNA is indeed encapsidated and that nucleotide substitutions in the translational operator which reduce the tightness of the Coat Protein-operator interaction also reduce or abolish encapsidation of the hybrid RNA. When Coat Protein is expressed in excess compared to the operator-lacZ RNA, host RNAs are packaged as well. However, elevation of the level of operator-lacZ RNA relative to Coat Protein results in its selective encapsidation at the expense of cellular RNAs. Our results are consistent with the proposition that this single Protein-RNA interaction accounts both for translational repression and viral genome encapsidation.
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The RNA binding site of bacteriophage MS2 Coat Protein.
The EMBO journal, 1993Co-Authors: David S. PeabodyAbstract:The Coat Protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of Coat Protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant Coat Proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of Protein folding or stability defects. Each of the variant Coat Proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of Coat Protein reveals that amino acid residues on three adjacent strands of the Coat Protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral Coat.