The RNA-binding Site of Bacteriophage Qβ Coat Protein

Lim, Francis; Spingola, Marc; Peabody, David S.

doi:10.1074/jbc.271.50.31839

Cited by 51 publications

(55 citation statements)

References 18 publications

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“…This experiment was performed twice with similar results. act as translation repressors that recognize similar RNA hairpins differing primarily in the 4-base loop sequence (see Lim et al (1994)). Discernment of the different RNA loop sequences by MS2 and GA coat proteins was attributed to a single amino acid residue (Lim et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

“…act as translation repressors that recognize similar RNA hairpins differing primarily in the 4-base loop sequence (see Lim et al (1994)). Discernment of the different RNA loop sequences by MS2 and GA coat proteins was attributed to a single amino acid residue (Lim et al, 1994). Thus, while the relatedness of IRP-1 and IRP-2 (Rouault et al, 1992) may account for their shared capacity to bind a wild-type IRE, it should prove at least equally as interesting to learn the amino acid differences which decide the individual specificities of the two IRPs.…”

Section: Discussionmentioning

confidence: 99%

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Iron Regulatory Proteins 1 and 2 Bind Distinct Sets of RNA Target Sequences

Henderson¹,

Menotti²,

Kühn³

1996

Journal of Biological Chemistry

104

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Iron regulatory proteins (IRPs) 1 and 2 bind with equally high affinity to iron-responsive element (IRE) RNA stem-loops located in mRNA untranslated regions and, thereby, post-transcriptionally regulate several genes of iron metabolism. In this study we define the RNA-binding specificities of mouse IRP-1 and IRP-2. By screening loop mutations of the ferritin H-chain IRE, we show that both IRPs bind well to a large number of IRE-like sequences. More significantly, each IRP was found to recognize a unique subset of IRE-like targets. These IRP-specific groups of IREs are distinct from one another and are characterized by changes in certain paired (IRP-1) or unpaired (IRP-2) loop nucleotides. We further demonstrate the application of such sequences as unique probes to detect and distinguish IRP-1 from IRP-2 in human cells, and observe that the IRPs are regulated similarly by iron and reducing agents in human and rodent cells. Importantly, the ability of each IRP to recognize an exclusive subset of IREs was conserved between species. These findings suggest that IRP-1 and IRP-2 may each regulate unique mRNA targets in vivo, possibly extending their function beyond the regulation of intracellular iron homeostasis.The regulation of cellular iron homeostasis is under the post-transcriptional control of iron regulatory protein-1 (IRP-1), 1 a cytoplasmic RNA-binding protein with specificity for mRNA stem-loop structures known as iron-responsive elements (IREs) (reviewed by Klausner et al. (1993) and Kü hn (1994)). IRP-1, formerly referred to as IRE-binding protein Leibold and Munro, 1988), iron regulatory factor (IRF; Mü llner et al., 1989), or ferritin repressor protein (Walden et al., 1988), has been identified as the cytosolic counterpart of the citric acid cycle enzyme, aconitase (Hentze and Argos, 1991;Rouault et al., 1991;Kaptain et al., 1991;Haile et al., 1992a;Kennedy et al., 1992). IRP-1 is now regarded as a bi-functional "sensor" of iron, switching between RNA binding and enzymatic activities depending on cellular iron status (Haile et al., 1992a(Haile et al., , 1992bConstable et al., 1992; EmeryGoodman et al., 1993;Basilion et al., 1994). In iron-depleted cells, IRP-1 inhibits translation of ferritin and erythroid 5-aminolevulinic acid synthase mRNAs by binding to IREs located in their 5Ј-untranslated region (UTR) (Aziz and Munro, 1987;Hentze et al., 1987;Bhasker et al., 1993;Melefors et al., 1993). Binding of IRP-1 to a cluster of five IREs in the 3Ј-UTR of transferrin receptor mRNA stabilizes this transcript (Casey et al., 1988;Mü llner and Kü hn, 1988;Mü llner et al., 1989;Koeller et al., 1989). The net result of this RNA-protein interaction is thus increased cellular iron uptake and availability. The inverse effect ensues when iron is high, as IRP-1 no longer binds well to the IRE hairpins (reviewed by Kü hn (1994)).A second IRE-binding protein has been characterized in rodents Guo et al., 1994) and in human cells (Samaniego et al., 1994), and is now commonly referred to as IRP-2. IRP-2 is a 105-kDa protein...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Iron Regulatory Proteins 1 and 2 Bind Distinct Sets of RNA Target Sequences

Henderson¹,

Menotti²,

Kühn³

1996

Journal of Biological Chemistry

104

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“…Translational repression was assessed by the ability of coat proteins expressed from pCT119, pP7CTNcXb, and pQCT to inhibit the synthesis of ␤-galactosidase from pRZ5, pRZP7, and pRZQ5 (10,13). Assays of ␤-galactosidase were performed using the method described by Miller (16).…”

Section: Methodsmentioning

confidence: 99%

“…Mapping the RNA Binding Site of PP7 Coat Protein-We previously mapped amino acid residues contributing to the RNA binding sites of MS2 and Q␤ coat proteins by random mutagenesis, followed by selection of mutants that failed to repress ␤-galactosidase synthesis in the two-plasmid systems already described (9,10,13). A further screen for capsid assembly eliminated mutants whose repressor defects were consequences of failure to properly fold, and DNA sequence analysis identified the affected amino acid residues.…”

Section: Disaggregation Of Capsids and Refolding Of Pp7 Coat Protein mentioning

confidence: 99%

“…Therefore, our first task was to determine whether PP7 coat protein is indeed a translational repressor and to identify its RNA binding target. We had previously constructed two-plasmid systems for MS2, GA, and Q␤ in which coat protein expressed from one plasmid represses translation of a replicase-␤-galactosidase fusion protein expressed from a second plasmid (9,10,13). Inspection of the sequence of PP7 RNA reveals the potential RNA hairpin structure shown in Fig.…”

Section: Construction Of Plasmids For Overexpression Of Coat Protein mentioning

confidence: 99%

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Translational Repression and Specific RNA Binding by the Coat Protein of the Pseudomonas Phage PP7

Lim

Downey

Peabody

2001

Journal of Biological Chemistry

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PP7 is a single-strand RNA bacteriophage of Pseudomonas aeroginosa and a distant relative to coliphages like MS2 and Q␤. Here we show that PP7 coat protein is a specific RNA-binding protein, capable of repressing the translation of sequences fused to the translation initiation region of PP7 replicase. Its RNA binding activity is specific since it represses the translational operator of PP7, but does not repress the operators of the MS2 or Q␤ phages. Conditions for the purification of coat protein and for the reconstitution of its RNA binding activity from disaggregated virus-like particles were established. Its dissociation constant for PP7 operator RNA in vitro was determined to be about 1 nM. Using a genetic system in which coat protein represses translation of a replicase-␤-galactosidase fusion protein, amino acid residues important for binding of PP7 RNA were identified.The coat proteins of several single-strand RNA bacteriophages are known translational repressors. They shut off viral replicase synthesis by binding an RNA hairpin that contains the replicase ribosome binding site. Recent x-ray structure determination of RNA phages shows that homologies evident from comparisons of coat protein amino acid sequences are reflected in their tertiary structures (1-7). The coat protein dimer, which is both the repressor and the basic building block of the virus particle, consists of two intertwined monomers that together form a large ␤-sheet surface upon which the RNA is bound. Each of the coat proteins uses a common structural framework to bind different RNAs, thereby presenting an opportunity to investigate the basis of specific RNA-protein recognition. In previous work we characterized the RNA binding sites of MS2, GA, and Q␤ coat proteins (8 -10). Here we describe the RNA binding properties of the coat protein of PP7, an RNA bacteriophage of Pseudomonas aeroginosa whose coat protein shows only 13% amino acid sequence identity to that of MS2. We present the following findings. 1) The coat protein of PP7 is a translational repressor. 2) An RNA hairpin containing the PP7 replicase translation initiation site is specifically bound by PP7 coat protein both in vivo and in vitro, indicating that this structure represents the translational operator.3) The RNA binding site resides on the coat protein ␤-sheet. A map of this site is presented. EXPERIMENTAL PROCEDURESPlasmid Constructions-The PP7 coat sequence cloned on a plasmid was kindly provided to us by Gordon Garde. We amplified the coat sequence using polymerase chain reaction and a 5Ј-primer (5Ј-GGGTCTAGACGTTACAGCGACTACTGAAACGTAAG-3Ј) that introduced a XbaI site about 40 nucleotides upstream of the coat initiation codon, and a 3Ј primer (5Ј-GGGGGATCCATACACACGGGTACACCG-CAGGGCC-3Ј) that created a BamHI site a few nucleotides downstream of the stop codon. This and subsequent amplifications were conducted using Pfu DNA polymerase. After digestion with XbaI and BamHI, the fragment was cloned between the corresponding restriction sites within the polylinker of pUC119 (11...

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Critical Roles for Arginine 1061/1060 and Tyrosine 1057 inSaccharomyces cerevisiaeArginine-Specific Carbamoyl-Phosphate Synthetase

Lim

Powers‐Lee

1997

Archives of Biochemistry and Biophysics

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The RNA-binding Site of Bacteriophage Qβ Coat Protein

Cited by 51 publications

References 18 publications

Iron Regulatory Proteins 1 and 2 Bind Distinct Sets of RNA Target Sequences

Iron Regulatory Proteins 1 and 2 Bind Distinct Sets of RNA Target Sequences

Translational Repression and Specific RNA Binding by the Coat Protein of the Pseudomonas Phage PP7

Critical Roles for Arginine 1061/1060 and Tyrosine 1057 inSaccharomyces cerevisiaeArginine-Specific Carbamoyl-Phosphate Synthetase

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