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A Thain

Publications and source records attributed to A Thain.

5 recordsLinked to original sources
PubMed2001

A tetracycline-binding RNA aptamer.

Aptamers are perfect tools to study the interaction of small ligands with RNA. To study the mode of interaction of tetracycline with RNA, we isolated aptamers with high affinity to this antibiotic via in vitro selection. One of the selected aptamers, cb28, which has a comparable affinity to tetracycline as the small ribosomal subunit, was characterised in more detail. Cb28 binds only to typical tetracyclines, while atypical tetracyclines are not recognised. The hydroxyl group at position 6 is an essential determinant for recognition, while modifications at positions 4, 5 and 7 do not interfere with RNA binding. Binding of tetracycline to cb28 is magnesium dependent. The secondary structure of cb28 was determined by lead cleavage and DMS modification. Upon tetracycline binding, nucleotides in J2/3 and the P5 stem-loop are protected from cleavage by lead, indicating a conformational change in the RNA. This conformational change was confirmed by tetracycline dependent changes in the DMS modification pattern. Photo-induced affinity incorporation of tetracycline into cb28 resulted in a crosslink to position G76, a residue in L5. The mode of binding of tetracycline to the cb28 aptamer resembles its interaction with the primary binding site on the small ribosomal subunit.

Anti-Bacterial Agents↗
PubMed1997

DNA binding and bending by the human papillomavirus type 16 E2 protein. Recognition of an extended binding site.

The human papillomavirus (HPV) 16 E2 protein (hE2) binds to four sites present upstream of the P97 promoter and regulates transcription of the viral E6 and E7 oncogenes. We have determined the relative binding constants for the interaction of the full-length hE2 protein with these sites. Our results show that hE2 binds tightly to site 4, less tightly to sites 1 and 2, and weakly to site 3. Similar results have previously been obtained using a C-terminal fragment of the hE2 protein suggesting that the C-terminal domain is the sole determinant of DNA binding affinity and specificity. Using circular permutation assays we show that binding of the hE2 protein induces the formation of a significant DNA bend and that the hE2-induced DNA bend angle is the same at both tight and weak hE2-binding sites. An alignment of the four hE2-binding sites from the HPV 16 genome suggests that this protein recognizes an extended binding site when compared with the bovine papillomavirus E2 protein. Here we show that the hE2 protein binds tightly to sites containing an A:T or a G:C base pair at position 7 of its binding site but weakly to sites containing either C:G or T:A at this position. Using site-directed mutagenesis we demonstrate that an arginine at position 304 of the hE2 protein is responsible for the recognition of specific base pairs at this position.

Binding Sites↗
PubMed1996

CpG methylation directly inhibits binding of the human papillomavirus type 16 E2 protein to specific DNA sequences.

CpG methylation of the human papillomavirus upstream regulatory region has previously been shown to reduce virus promoter activity. Here, we demonstrate that methylation of the CpG dinucleotides contained within the binding site of the human papillomavirus type 16 E2 protein has a direct effect on the interaction of this protein with DNA. Methylation of both CpG dinucleotides within the E2 site abolishes the binding of E2.

DNA, Viral↗