Analysis of HutP-dependent transcription antitermination in the Bacillus subtilis hut operon: identification of HutP binding sites on hut antiterminator RNA and the involvement of the N-terminus of HutP in binding of HutP to the antiterminator RNA.
We investigated HutP-dependent transcription antitermination of the Bacillus subtilis hut operon. In vitro transcription assays with the B. subtilissigmaA-containing RNA polymerase indicated that HutP inhibits transcription termination at the internal terminator by binding to the antiterminator on hut mRNA in the presence of histidine. Ethylnitrosourea modification interference assays and mutational analyses of the interference sites showed that interaction of HutP with a region containing three UAG trinucleotide sequences, which is located on top of the antiterminator structure, is critical for hut antitermination in vivo. Results from kinetic analysis of binding of HutP to RNA containing various portions of the antiterminator sequences indicated that secondary structure is required for binding of HutP to the region containing three UAG triplets in the antiterminator. The in vivo HutP antiterminator activity was reduced by the mutations in the N-terminal region of HutP. The HutP variants with H4A, R7A, I9A and Q26A mutations exhibited reduced binding affinities to the antiterminator RNA in vitro. A 25-mer peptide consisting of amino acid residues 2-26 of HutP bound to the antiterminator RNA. These results indicated that the N-terminus of HutP is involved in binding of HutP to the antiterminator RNA.
Oda
Masanao
M
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba City, Ibaraki, Japan. masa-oda@aist.go.jp
Kobayashi
Nobuhiro
N
Fujita
Masaya
M
Miyazaki
Yuusuke
Y
Sadaie
Yoshito
Y
Kurusu
Yasurou
Y
Nishikawa
Satoshi
S
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004252:DNA Mutational Analysis; D005798:Genes, Bacterial; D006639:Histidine / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009690:Nucleic Acid Conformation; D009876:Operon; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C064872:antiterminator proteins, Bacteria; D006639:Histidine