The E26 avian retrovirus causes an acute leukemia in chickens and transforms both myeloid and erythroid cells. The virus encodes a 135 kDa fusion protein which contains amino acid sequences derived from the viral Gag protein and the two cellular transcription factors c‐Myb and c‐Ets‐1p68. Previously we have shown that like v‐myb, v‐ets on its own is also active in transformation, but only within the erythroid lineage. To understand better the mechanisms involved in the oncogenic activation of c‐Ets‐1p68, we used the polyoma PEA3 element, a known Ets binding site, to compare the sequence‐specific DNA binding and transactivating properties of v‐Ets and c‐Ets‐1p68. Using Ets protein synthesized in rabbit reticulocyte lysate in gel retardation assays, we detected little binding of c‐Ets‐1p68 to an oligonucleotide containing the PEA3 motif whereas v‐Ets bound strongly. However, in transient cotransfection assays in chicken embryo fibroblasts both c‐Ets‐1p68 and v‐Ets transactivated transcription from a heterologous promoter linked to PEA3 elements. Interestingly, fragments of c‐Ets‐1p68 with strong DNA binding activity could be produced by limited proteolysis, indicating that the DNA binding domain is repressed within the full‐length molecule. By deletion mapping the DNA binding domain was localized to the most highly conserved region of the Ets‐related proteins known as the ETS domain. The C‐terminus as well as a region in the middle of the polypeptide chain are involved in repression of DNA binding in c‐Ets‐1p68. Significantly, v‐Ets contains a 16 amino acid substitution at the C‐terminus. Our results suggest that intramolecular repression of DNA binding is a regulatory mechanism in c‐Ets‐1p68 which is lost in v‐Ets.

The coat proteins of different single-strand RNA phages use a common protein tertiary structural framework to recognize different RNA hairpins and thus offer a natural model for understanding the molecular basis of RNA-binding specificity. Here we describe the RNA structural requirements for binding to the coat protein of bacteriophage PP7, an RNA phage of Pseudomonas. Its recognition specificity differs substantially from those of the coat proteins of its previously characterized relatives such as the coliphages MS2 and Qbeta. Using designed variants of the wild-type RNA, and selection of binding-competent sequences from random RNA sequence libraries (i.e. SELEX) we find that tight binding to PP7 coat protein is favored by the existence of an 8 bp hairpin with a bulged purine on its 5' side separated by 4 bp from a 6 nt loop having the sequence Pu-U-A-G/U-G-Pu. However, another structural class possessing only some of these features is capable of binding almost as tightly.

The coat protein of bacteriophage MS2 functions as a symmetric dimer to bind an asymmetric RNA hairpin. This implies the existence of two equivalent RNA binding sites related to one another by a 2-fold symmetry axis. In this view the symmetric binding site defined by mutations conferring the repressor-defective phenotype is a composite picture of these two asymmetric sites. In order to determine whether the RNA ligand interacts with amino acid residues on both subunits of the dimer and in the hope of constructing a functional map of the RNA binding site, we performed heterodimer complementation experiments. Taking advantage of the physical proximity of their N- and C-termini, the two subunits of the dimer were genetically fused, producing a duplicated coat protein which folds normally and allows the construction of the functional equivalent of obligatory heterodimers containing all possible pairwise combinations of the repressor-defective mutations. The restoration of repressor function in certain heterodimers shows that a single RNA molecule interacts with both subunits of the dimer and allows the construction of a functional map of the binding site.

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...

The coat protein of the RNA bacteriophage MS2 is a specific RNA binding protein that represses translation of the viral replicase gene during the infection cycle. As an approach to characterizing the RNA-binding site of coat protein we have isolated a series of coat mutants that suppress the effects of a mutation in the translational operator. Each of the mutants exhibits a super-repressor phenotype, more tightly repressing both the mutant and wild-type operators than does the wild-type protein. The variant coat proteins were purified and subjected to filter binding assays to determine their affinities for the mutant and wild-type operators. Each protein binds the operators from 3 to 7.5-fold more tightly than normal coat protein. The amino acid substitutions seem to extend the normal binding site by introducing new interactions with RNA.