Translation initiation factor IF1 is a small, essential and ubiquitous protein factor encoded by a single infA gene in bacteria. Although several important functions have been attributed to IF1, the precise reason for its indispensability is yet to be defined. It is known that IF1 binds to the ribosomal A-site during initiation, where it primarily contacts ribosomal RNA (rRNA) and induces large scale conformational changes in the small ribosomal subunit. To shed more light on the function of IF1 and its interaction with the ribosome, we have employed a genetic approach to elucidate structure-function interactions between IF1 and rRNA. A selection has been used to isolate second site suppressor mutations in rRNA that restore the growth of a cold sensitive mutant IF1 with an arginine to leucine substitution in position 69 (R69L). This yielded two classes of suppressors – one class that mapped to the processing stem of 23S rRNA – a transient structure important for proper maturation of 23S rRNA; and the other class to the functional sequence of 16S rRNA. Suppressor mutations in the processing stem of 23S rRNA were shown to disrupt efficient processing of 23S rRNA. In addition, we report that at least one of the manifestations of cold sensitivity associated with the mutant IF1 is at the level of ribosomal subunit association. These results led to a model whereby the cold sensitive R69L mutant IF1 results in aberrant ribosomal subunit association properties, while the 23S processing stem mutations indirectly suppress this effect by decreasing the pool of mature 50S subunits available for association. Spontaneous suppressor mutations in 16S rRNA were diverse in position and phenotypic properties, but all mutations affected ribosomal subunit association, in most cases by directly decreasing the affinity of the 30S for 50S subunits. Site directed mutagenesis of select positions in 16S rRNA yielded additional suppressor mutations that were localized to the mRNA and streptomycin binding sites on the small ribosomal subunit. We suggest that the 16S rRNA suppressors occur in positions that affect the conformational dynamics brought about by IF1. Taken together, this work indicates that the major function of IF1 is the modulation of ribosomal subunit association brought about through conformational changes of the 30S subunit. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:su-75363 |
| Date | January 2012 |
| Creators | Belotserkovsky, Jaroslav |
| Publisher | Stockholms universitet, Institutionen för genetik, mikrobiologi och toxikologi, Stockholm : Department of Genetics, Microbiology and Toxicology, Stockholm University |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Doctoral thesis, comprehensive summary, info:eu-repo/semantics/doctoralThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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