Functional analysis of the 14 kDa protein of insertion sequence 2

Shiau Ting Hu, Jar Hsien Hwang, Lim Chee Lee, Chao-Hung Lee, Pei Lun Li, Yueh Chun Hsieh

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

The IS2 sequence encodes five open reading frames (ORF1 to ORF5) that are greater than 150 nucleotides each. Only one protein of 14 kDa was detected when the expression of IS2 genes was examined in minicells. This 14 kDa protein was referred to as InsA in this study and was determined to be encoded by ORF1. A sixfold decrease in IS2 transposition frequency was observed when insA was overexpressed. DNA footprinting results indicated that InsA binds to the sequence 5'-TAAATAA-3' located at IS2 nucleotide numbers 1286 to 1292. (The IS2 right terminal repeat spans nucleotides 1290 to 1331.) This InsA binding sequence is situated 4 bp upstream from the putative '-10' sequence of the insA promoter that overlaps the right terminal repeat of IS2. The presence of a promoter located in this region was demonstrated by the ability of a DNA fragment containing the right terminal repeat to drive the expression of a promoterless lacZ gene. The transcription of insA was determined to start at the A residue located at nucleotide number 1268. With the same insA promoter-lacZ fusion construct, overexpression of insA in the same cell was found to decrease the β-galactosidase activity. The results of this study suggest that InsA affects IS2 transposition by regulating the transcription of IS2 genes.

Original languageEnglish
Pages (from-to)503-513
Number of pages11
JournalJournal of Molecular Biology
Volume236
Issue number2
DOIs
StatePublished - 1994

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Insertional Mutagenesis
Terminal Repeat Sequences
Nucleotides
Proteins
DNA Footprinting
Galactosidases
Lac Operon
Open Reading Frames
Gene Expression
DNA
Genes

Keywords

  • InsA binding sequence
  • IS2
  • IS2 InsA protein
  • transposable element
  • transposition

ASJC Scopus subject areas

  • Virology

Cite this

Functional analysis of the 14 kDa protein of insertion sequence 2. / Hu, Shiau Ting; Hwang, Jar Hsien; Lee, Lim Chee; Lee, Chao-Hung; Li, Pei Lun; Hsieh, Yueh Chun.

In: Journal of Molecular Biology, Vol. 236, No. 2, 1994, p. 503-513.

Research output: Contribution to journalArticle

Hu, Shiau Ting ; Hwang, Jar Hsien ; Lee, Lim Chee ; Lee, Chao-Hung ; Li, Pei Lun ; Hsieh, Yueh Chun. / Functional analysis of the 14 kDa protein of insertion sequence 2. In: Journal of Molecular Biology. 1994 ; Vol. 236, No. 2. pp. 503-513.
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