Characterization of conserved bases in 4.5S RNA of Escherichia coli by construction of new F' factors

Abstract

To more clearly understand the function of conserved bases of 4.5S RNA, the product of the essential ffs gene of Escherichia coli, and to address conflicting results reported in other studies, we have developed a new genetic system to characterize ffs mutants. Multiple ffs alleles were generated by altering positions that correspond to the region of the RNA molecule that interacts directly with Ffh in assembly of the signal recognition particle. To facilitate characterization of the ffs mutations with minimal manipulation, recombineering was used to construct new F' factors to easily move each allele into different genetic backgrounds for expression in single copy. In combination with plasmids that expressed ffs in multiple copy numbers, the F' factors provided an accurate assessment of the ability of the different 4.5S RNA mutants to function in vivo. Consistent with structural analysis of the signal recognition particle (SRP), highly conserved bases in 4.5S RNA are important for binding Ffh. Despite the high degree of conservation, however, only a single base (C62) was indispensable for RNA function under all conditions tested. To quantify the interaction between 4.5S RNA and Ffh, an assay was developed to measure the ability of mutant 4.5S RNA molecules to copurify with Ffh. Defects in Ffh binding correlated with loss of SRP-dependent protein localization. Real-time quantitative PCR was also used to measure the levels of wild-type and mutant 4.5S RNA expressed in vivo. These results clarify inconsistencies from prior studies and yielded a convenient method to study the function of multiple alleles.

FIG. 1.

Summary of the genetic system used for characterizing ffs mutants. (A) Recombineering was used to construct a series of F′ factors expressing different ffs alleles. As explained in Materials and Methods, galK⁺ was first incorporated into the F′ plasmid by recombineering using homology to lacA carried by F′ lac⁺ pro⁺. Next, individual ffs alleles are introduced to the F′ elements by selecting against galK⁺ by resistance to 2-DOG. (B) JP2005 has a deletion of the chromosomal copy of ffs and carries a complementing copy of ffs on a plasmid that is temperature sensitive for replication. F′ plasmids or ColE1-derivative plasmids bearing mutant alleles of ffs were introduced by conjugation or transformation, respectively. The ability of the ffs alleles to support growth of JP2005 was determined by comparing the efficiency of plating at 30°C and 42°C, the nonpermissive temperature of the temperature-sensitive plasmid.

FIG. 2.

Summary of 4.5S RNA mutants. The primary sequence and predicted secondary structure of 4.5S RNA is shown at the top. Helix 8, the region bound by Ffh, is underlined. The mutations generated in this study are indicated by arrows. The degree of conservation of each base among SRP RNAs from all three domains of life is represented by the size of the base, with the largest sizes representing the most highly conserved. The consensus sequence of SRP RNA is shown at the bottom, where the largest-sized bases are also most highly conserved. The numbers represent the positions of the bases in E. coli 4.5S RNA. The degree of base conservation and the consensus sequence were from the SRP database (1).

FIG. 3.

Growth of ffs mutants. Cultures of JP2005 transformed with plasmids expressing different ffs alleles in multiple copy numbers were grown as described in Materials and Methods. Each ffs allele is specified in the columns on the left. Images represent identical colonies formed after an initial incubation at 42°C followed by further incubation at 30°C, as indicated on the right of each column.

FIG. 4.

Detection of biotinylated FtsQ. The ffs mutants shown were tested to determine the extent of biotinylation of the SRP-dependent protein FtsQ. (Top) Biotinylated FtsQ as detected by decoration with streptavidin-horseradish peroxidase conjugate. (Bottom) FtsQ as detected by Western blot analysis using antibody specific for the V5 epitope. A plus sign indicates ffs⁺, and other alleles are as designated.