Alterations in the two globular domains or in the connecting alpha-helix of bacterial ribosomal protein L9 induces +1 frameshifts

Abstract

The ribosomal 50S subunit protein L9, encoded by the gene rplI, is an elongated protein with an alpha-helix connecting the N- and C-terminal globular domains. We isolated rplI mutants that suppress the +1 frameshift mutation hisC3072 in Salmonella enterica serovar Typhimurium. These mutants have amino acid substitutions in the N-terminal domain (G24D) or in the C-terminal domain (I94S, A102D, G126V, and F132S) of L9. In addition, different one-base deletions in rplI altered either the final portion of the C terminus or removed the C-terminal domain with or without the connecting alpha-helix. An alanine-to-proline substitution at position 59 (A59P), which breaks the alpha-helix between the globular domains, induced +1 frameshifting, suggesting that the geometrical relationship between the N and C domains is important to maintain the reading frame. Except for the alterations G126V in the C terminus and A59P in the connecting alpha-helix, our results confirm earlier results obtained by using the phage T4 gene 60-based system to monitor bypassing. The way rplI mutations suppress various frameshift mutations suggests that bypassing of many codons from several takeoff and landing sites occurred instead of a specific frameshift forward at overlapping codons.

FIG. 1.

Alterations in protein L9. The presented protein structure shows L9 from Bacillus (18). The N- and C-terminal globular domains are to the left and right, respectively, with the connecting α-helix in between. Since protein L9 in Bacillus and Salmonella is conserved, the indicated mutation sites are numbered according to L9 of Salmonella. The amino acid substitutions are in boldface; the frameshift mutations are in italics. An asterisk with the amino acid symbol marks the codon in which it occurred.

FIG. 2.

pUST274 plasmid construct carrying the +1 frameshift site. (A) The malE gene is in the +1 frame relative to the gst gene. The PreScission protease recognition site in the 3′ terminus of gst sequence is indicated as a black box, the first stop codon (TAA) encountered in the 0 frame in malE is marked by an asterisk, and the His₆ tag is indicated as a white box. The total length of the frameshift window generating the GST-MalE fusion is: AAG-TAT-ATA-GCA-TGG-CCT-TTG-CAG-GGC-TGG-CAA-GCC-ACG-TTT-GGT-GGT-GGC-GAC-CAT-CCT-CCA-AAA-TCG-GAT-CTG-GAA-GTT-CTG-TTC-CAG-GGT-CCA-CTC-GGG-ATC-CCG-GGG-GAA-AGA-CGC-CAT-TCT-CTA-CTG- TCC-GAA-TTC-CCA-ACT-GAA-AAT-CGA-AGA-AGG-TAA. The UAG and UAA stop codons in +1 and 0 frame, respectively, are in boldface. The underlined portion is the sequence corresponding to the N-terminal sequence generated following digestion by PreScission protease as also shown in the panel. The BamHI and EcoRI sites used for inserting the desired frameshift site are indicated in italics. (B) Frameshifting (FS) and termination (Term) products from the wild type and rplI11<>Cm^r mutant separated on a sodium dodecyl sulfate gel and detected by anti-GST antibodies by Western blot analysis.