Saturation mutagenesis of 5S rRNA in Saccharomyces cerevisiae.

rRNAs are the central players in the reactions catalyzed by ribosomes, and the individual rRNAs are actively involved in different ribosome functions. Our previous demonstration that yeast 5S rRNA mutants (called mof9) can impact translational reading frame maintenance showed an unexpected function for this ubiquitous biomolecule. At the time, however, the highly repetitive nature of the genes encoding rRNAs precluded more detailed genetic and molecular analyses. A new genetic system allows all 5S rRNAs in the cell to be transcribed from a small, easily manipulated plasmid. The system is also amenable for the study of the other rRNAs, and provides an ideal genetic platform for detailed structural and functional studies. Saturation mutagenesis reveals regions of 5S rRNA that are required for cell viability, translational accuracy, and virus propagation. Unexpectedly, very few lethal alleles were identified, demonstrating the resilience of this molecule. Superimposition of genetic phenotypes on a physical map of 5S rRNA reveals the existence of phenotypic clusters of mutants, suggesting that specific regions of 5S rRNA are important for specific functions. Mapping these mutants onto the Haloarcula marismortui large subunit reveals that these clusters occur at important points of physical interaction between 5S rRNA and the different functional centers of the ribosome. Our analyses lead us to propose that one of the major functions of 5S rRNA may be to enhance translational fidelity by acting as a physical transducer of information between all of the different functional centers of the ribosome.

Ribosomal protein L5 helps anchor peptidyl-tRNA to the P-site in Saccharomyces cerevisiae.

Our previous demonstration that mutants of 5S rRNA called mof9 can specifically alter efficiencies of programmed ribosomal frameshifting (PRF) suggested a role for this ubiquitous molecule in the maintenance of translational reading frame, though the repetitive nature of the 5S rDNA gene (>100 copies/cell) inhibited more detailed analyses. However, given the known interactions between 5S rRNA and ribosomal protein L5 (previously called L1 or YL3) encoded by an essential, single-copy gene, we monitored the effects of a series of well-defined rpl5 mutants on PRF and virus propagation. Consistent with the mof9 results, we find that the rpl5 mutants promoted increased frameshifting efficiencies in both the -1 and +1 directions, and conferred defects in the ability of cells to propagate two endogenous viruses. Biochemical analyses demonstrated that mutant ribosomes had decreased affinities for peptidyl-tRNA. Pharmacological studies showed that sparsomycin, a peptidyltransferase inhibitor that specifically increases the binding of peptidyl-tRNA with ribosomes, was antagonistic to the frameshifting defects of the most severe mutant, and the extent of sparsomycin resistance correlated with the severity of the frameshifting defects in all of the mutants. These results provide biochemical and physiological evidence that one function of L5 is to anchor peptidyl-tRNA to the P-site. A model is presented describing how decreased affinity of ribosomes for peptidyl-tRNA can affect both -1 and +1 frameshifting, and for the effects of sparsomycin.

Ty1 retrotransposition and programmed +1 ribosomal frameshifting require the integrity of the protein synthetic translocation step.

Programmed ribosomal frameshifting is utilized by a number of RNA viruses to ensure the correct ratio of viral structural to enzymatic proteins for viral particle assembly. Altering frameshifting efficiencies upsets this ratio, inhibiting virus propagation. Two yeast viruses that induce host cell ribosomes to shift translational reading frame were used as tools to explore the interactions between viruses and host cellular protein synthetic machinery. Previous studies showed that the ribosome-inactivating protein pokeweed antiviral protein specifically inhibited propagation of the Ty1 retrotransposable element of yeast as a consequence of inhibition of programmed +1 ribosomal frameshifting. Here, complementary genetic and pharmacological approaches were employed to test whether inhibition of Ty1 retrotransposition is a general feature of alterations in the translocation step of elongation and +1 frameshifting. The results demonstrate that cells harboring a variety of mutant alleles of two host-encoded proteins that are involved in translocation, eukaryotic elongation factor-2 and the ribosome-associated protein RPP0, have Ty1 propagation defects. We also show that sordarin, a fungus-specific inhibitor of eEF-2 function, specifically inhibits programmed +1 ribosomal frameshifting and Ty1 retrotransposition. These findings serve to link inhibition of Ty1 retrotransposition and +1 frameshifting to changes in the translocation step of elongation.

Mathematical modelling of morphogenesis in fungi: a key role for curvature compensation ('autotropism') in the local curvature distribution model.

The assumption that the mushroom stem has the ability to undergo autonomic straightening enables a mathematical model to be written that accurately mimics the gravitropic reaction of the stems of Coprinus cinereus. The straightening mechanism is called curvature compensation here, but is equivalent to the 'autotropism' that often accompanies the gravitropic reactions of axial organs in plants. In the consequently revised local curvature distribution model, local bending rate is determined by the difference between the 'bending signal' (generated by gravitropic signal perception systems) and the 'straightening signal' (proportional to the local curvature at the given point). The model describes gravitropic stem bending in the standard assay with great accuracy but has the virtue of operating well outside the experimental data set used in its derivation. It is shown, for example, that the mathematical model can be fitted to the gravitropic reactions of stems treated with metabolic inhibitors by a change of parameters that parallel the independently derived physiological interpretation of inhibitor action. The revised local curvature distribution model promises to be a predictive tool in the further analysis of gravitropism in mushrooms.

Spatial organization of the gravitropic response in plants: applicability of the revised local curvature distribution model to Triticum aestivum coleoptiles.

The revised local curvature distribution model, which provides accurate computer simulations of the gravitropic response of mushroom stems, was found to produce accurate simulations of the gravitropic reaction of wheat (Triticum aestivum) coleoptiles. The key feature of the mathematical model that enables it to approach universality of application is the assumption that the stem has an autonomic straightening reaction (curvature compensation or 'autotropism'). In the model, the local bending rate for any segment of the organ is determined by the difference between the 'bending signal' (generated by the gravitropic signal perception system) and a 'straightening signal' (which is proportional to the local curvature of the segment). The model reveals three major differences between the gravitropic reactions of wheat coleoptiles and Coprinus mushroom stems. First, in Coprinus, the capacity for autonomic straightening is much more concentrated in the apical region of the stem. Second, local perception of the gravitropic signal, which is necessary for exact simulation in Coprinus, is not needed in wheat coleoptiles (the corresponding constant in the model can be set to zero). Third, the transmission rate of the gravitropic signal is about seven times faster in wheat coleoptiles than in the mushroom stem. Thus, we demonstrate that a single model, depending on the values given to its parameters, is able to simulate the spatial organization of the gravitropic reaction of wheat coleoptiles and Coprinus mushroom stems. The model promises to be a valuable predictive tool in guiding future research into the gravitropic reaction of axial organs of all types.

Mathematical modelling of morphogenesis in fungi: spatial organization of the gravitropic response in the mushroom stem of Coprinus cinereus.

The purpose of this work was to establish how the distribution of local curvatures changed during the mushroom stem gravitropic reaction and to suggest a suitable mathematical model based on these new data. The gravitropic bending of base- and apex-pinned Coprinus cinereus (Fries) S. F. Gray stems was recorded on videotapes. The images were captured from the tapes after each 10 min, rotated by 45 degrees and transformed into tables of changing co-ordinates of points for each stem. The non-linear regression of these points was performed using Legendre polynomials. From the resulting equations the patterns of changing local curvature for 50 subsections per stem during 400 min of gravitropic reaction were calculated. It was observed that base-pinned stems first bent from the apex, but later the curvature of this part decreased, and in the late stages the apex became nearly completely straight again. Subsections, located about one third of stem length from the base determined the main part of the final curvature. The free basal part of the apex-pinned stems bent upward and after a certain bending time also began to straighten. However, this process started significantly later and was weaker. Bending of the subsections close to the pinned apex did not stop when they reached the vertical position, and the final angle of gravitropic curvature could exceed 180 degrees. Plotting various functions of local bending speed and its derivatives against each other and against local angle indicated that, if the hypothetical signal about reorientation arises in the apex, its propagation towards the base did not follow simple wave or simple diffusion laws. The importance of the local angle of all subsections both for signal origin and transmission was established and a signal transmission equation, involving local angle of each subsection, was derived. After creating a suitable program this partial differential equation was solved numerically. The generated shapes of the bending stem coincided in high degree with experimentally observed images.

The K2-type killer toxin- and immunity-encoding region from Saccharomyces cerevisiae: structure and expression in yeast.

The cDNA copies of M2-1, the larger heat-cleavage product of M2 double-stranded (ds) RNA, have been synthesized, cloned, sequenced and expressed in yeast. This sequence, in combination with the known terminal sequence of M2-1 dsRNA, identifies a translation reading frame for a 362-amino-acid protein of 38.7 kDa, similar in size to the one of several protein species produced from M2-1 dsRNA in vitro translation. The expression of this cDNA clone in yeast confers both killer and immunity phenotypes.

'Red pigment' from ADE-2 mutants of S. cerevisiae prevents DNA cleavage by restriction endonucleases.

Protection of DNA from cleavage by restriction endonucleases EcoRI, HindIII, BamHI, and Bg/II with red pigment, produced by ADE-2 mutants of Saccharomyces cerevisiae is demonstrated. Purification of yeast DNA from pigment can be achieved by chromatography on hydroxyapatite columns.

A model for the monocular line orientation analyzer.

A model for monocular line perception by human Ss is based on three basic assumptions: (a) the line's inclination is coded by the maximally excited orientation detector's number; (b) the inclination of the perceived line is equivocally determined by the excitation vector in the subjective space; (c) the analyzer has a maximum differential sensitivity over the whole range of the line inclinations. This simple model for the line inclination analyzer, taking into account the optimization of its sensitivity, provides incomplex explanations for a wide range of psychophysical and neurophysiological data obtained from human and animal experiments.