The SELEX method of in vitro selection was used to isolate RNAs that bind the RB69 RegA translational repressor protein immobilized on Ni-NTA agarose. After five rounds of SELEX, the pool of selected RNA displayed striking sequence uniformity: UAAUAAUAAUAAUA was clearly enriched in the 14 nucleotides that underwent selection. Individual, cloned molecules displayed a repeating (UAA) sequence, with only two RNAs having a 3' AUG. Removing the 3' AUG slightly reduced binding in gel shift assays, moving the AUG 5' proximal of the (UAA) slightly improved binding, but (UAA)4 alone still bound the purified protein. Dissociation constants showed that RNA shortened to (UAA)3 and (UAA)2 also retained binding, whereas cytosine clearly prevented binding by RB69 RegA. Scanning of RB69 gene starts and ends with an RB69 RegA SELEX information weight matrix yielded 21 sequences as potential RegA sites. One site, on the mRNA for the pentameric (4:1) phage gp44/62 DNA polymerase clamp loader complex, has the RB69 gene 44 stop codon and 3'-adjacent gene 62 initiation codon in a sequence (GAAAUAAUAUG) that is similar to in vitro selected RNA and was shown to bind RB69 RegA. Sequences between the Shine-Dalgarno and initiation codon, which frequently contain a UAA stop codon of a 5'-adjacent gene, appear to be preferred RB69 RegA binding sites.
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In vitro selection of phage RB69 RegA RNA binding sites yields UAA triplets.
Virology
May 2005
Department of Microbiology, North Carolina State University, Raleigh, NC 27695-7615, USA.
The SELEX method of in vitro selection was used to isolate RNAs that bind the RB69 RegA translational repressor protein immobilized on Ni-NTA agarose. After five rounds of SELEX, the pool of selected RNA displayed striking sequence uniformity: UAAUAAUAAUAAUA was clearly enriched in the 14 nucleotides that underwent selection. Individual, cloned molecules displayed a repeating (UAA) sequence, with only two RNAs having a 3' AUG.
View Article and Find Full Text PDFRegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities.
Nucleic Acids Res
March 2001
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.
The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays.
View Article and Find Full Text PDFRNA-binding properties of in vitro expressed histidine-tagged RB69 RegA translational repressor protein.
Anal Biochem
April 1999
Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27696-7615, USA.
To facilitate RNA-binding studies of the phage RB69 RegA translational repressor protein, regA was configured to add six histidines to the carboxyl end of the protein. In vitro transcription-translation from the T7 promoter on plasmid pSA1 yielded a RegA69-His6 protein that binds nickel-Sepharose and elutes with 0.5 M imidazole.
View Article and Find Full Text PDFRetention of replication fidelity by a DNA polymerase functioning in a distantly related environment.
Proc Natl Acad Sci U S A
July 1997
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, USA.
The primary structures of the replicative DNA polymerases (gp43s) of bacteriophage T4 and its distant phylogenetic relative RB69 are diverged, retaining only 61% identity and 74% similarity. Nevertheless, RB69 gp43 substitutes effectively for T4 gp43 in T4 DNA replication in vivo. We show here that RB69 gp43 replicates T4 genomes in vivo with a fidelity similar to that achieved by T4 gp43.
View Article and Find Full Text PDFEvolution of RNA-binding specificity in T4 DNA polymerase.
J Biol Chem
July 1997
Department of Biochemistry, SL43, Tulane University School of Medicine, New Orleans, Louisana 70112, USA.
DNA polymerase of phage T4 (T4 gp43), an essential component of the T4 DNA replicase, is a multifunctional single-chained (898-amino acid) protein that catalyzes the highly accurate synthesis of DNA in phage replication. The enzyme functions both as a DNA-binding replication protein and as a sequence-specific RNA-binding autogenous translational repressor. We have utilized a phylogenetic approach to study the relationships between the two nucleic acid-binding functions of the protein.
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