Four Inteins and Three Group II Introns Encoded in a Bacterial Ribonucleotide Reductase Gene

Xiang-Qin Liu,Jing Yang,Qing Meng

doi:10.1074/jbc.m309575200

Abstract

A bacterial ribonucleotide reductase gene was found to encode four inteins and three group II introns in the oceanic N2-fixing cyanobacterium Trichodesmium erythraeum. The 13,650-bp ribonucleotide reductase gene is divided into eight extein- or exon-coding sequences that together encode a 768-amino acid mature ribonucleotide reductase protein, with 83% of the gene sequence encoding introns and inteins. The four inteins are encoded on the second half of the gene, and each has conserved sequence motifs for a protein-splicing domain and an endonuclease domain. These four inteins, together with known inteins, define five intein insertion sites in ribonucleotide reductase homologues. Two of the insertion sites are 10 amino acids apart and next to key catalytic residues of the enzyme. Protein-splicing activities of all four inteins were demonstrated in Escherichia coli. The four inteins coexist with three group II introns encoded on the first half of the same gene, which suggests a breakdown of the presumed barrier against intron insertion in this bacterial conserved protein-coding gene.

Highlights

Inteins are intervening protein sequences embedded in precursor proteins and catalyze a protein-splicing reaction that excises the intein and joins the flanking sequences (N- and C-exteins) with a peptide bond [1,2,3,4]
The mature ribonucleotide reductase (RIR) sequence of T. erythraeum is 69% identical and 80% similar to its homologue in the cyanobacterium Nostoc sp. strain PCC7120 (Nsp), and this level of RIR sequence identity is normal among cyanobacterial species
The T. erythraeum RIR gene was found to encode four inteins and three group II introns, which predicts three RNA splicing events followed by four protein-splicing events in producing the mature ribonucleotide reductase (Fig. 1)

Summary

EXPERIMENTAL PROCEDURES

Gene Cloning and Sequence Analysis—GenBank searches, sequence alignments, and intron RNA folding were performed using the BLAST search program [30], the ClustalW program [31], and the MFOLD program [32], respectively. Protein-splicing Analysis in Escherichia coli Cells—To construct gene expression plasmids, intein coding sequences were inserted in the previously described pMST plasmid [33] between XhoI and AgeI sites, replacing the Ssp DnaB intein coding sequence of pMST. Protein production in E. coli cells, gel electrophoresis, and Western blot analysis were performed as described previously [33]. Isopropyl-1-thio-␤-D-galactopyranoside was added to a final concentration of 0.8 mM to induce production of the recombinant protein, and the induction was continued at 37 °C for 3 h or at.

Seven Intervening Sequences in a Bacterial Gene

RESULTS

DISCUSSION