Weakening the IF2-fMet-tRNA Interaction Suppresses the Lethal Phenotype Caused by GTPase Inactivation

Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome.

Propagation of seminal toxins through binary expression gene drives can suppress polyandrous populations

Gene drives can be highly effective in controlling a target population by disrupting a female fertility gene. To spread across a population, these drives require that disrupted alleles be largely recessive so as not to impose too high of a fitness penalty. We argue that this restriction may be relaxed by using a double gene drive design to spread a split binary expression system. One drive carries a dominant lethal/toxic effector alone and the other a transactivator factor, without which the effector will not act. Only after the drives reach sufficiently high frequencies would individuals have the chance to inherit both system components and the effector be expressed. We explore through mathematical modeling the potential of this design to spread dominant lethal/toxic alleles and suppress populations. We show that this system could be implemented to spread engineered seminal proteins designed to kill females, making it highly effective against polyandrous populations.

Inbreeding effects in Drosophila congenic strains: the influence of genetic background of different origin

Aim. To compare reproductive indices and stress resistance of Drosophila at outbreeding and inbreeding. Methods. Drosophila melanogaster congenic strains with incomplete development of the radial wing vein – radius incompletus – were used: the laboratory one and the strain, in which the mutation was placed into the genetic background of wild type strain, which originates from the natural population from radiation contaminated territory. Before the experiment strains have passed 65 generations of inbreeding. Viability (number of individuals, pupa stage mortality), dominant lethal mutations frequency and life span of imago at starvation were analysed. Results. After inbreeding, there was a decrease in the frequency of dominant lethal mutations and an increase in viability of the strain, which originates from the natural population, and a decrease of mortality at the pupal stage in both strains. Decreased life span of imago at starvation has been shown only for the inbred strain, which originates from the natural population. Conclusions. Inbreeding for 65 generations has no significant negative effect on reproductive indices; reduction of stress resistance during inbreeding has been shown only for the strain, which originates from the radiation contaminated territory. Keywords: Drosophila, viability, dominant lethal mutations, life span of imago at starvation, inbreeding.

Antimutagenic activity of probiotic drugs on the example of Drosophila

Probiotic preparations were obtained on the basis of cryopreserved forms of pure cultures of L. plantarum and B. subtilis using freeze drying. The antimutagenic activity of the obtained probiotic preparations was investigated in relation to 1%, 0.1% and 0.01% concentrations of cobalt sulfate using the example of dominant lethal mutations of Drosophila. A positive antimutagenic effect of probiotic preparations was shown in relation to 0.1% and 0.01% concentrations of CoSO4 with an exposure duration of at least 3 days.

Validation of Omega Subunit of RNA Polymerase as a Functional Entity

The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ββ’ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, β’, and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ββ’ subassembly. We observed that the interaction between α2ββ’ and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in rpoZ. We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code’s position in the reading frame of rpoZ plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ββ’. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the β’ subunit, and it is the same for both types of mutants.