Binding of wheat germ ribosomes to bisulfite-modified reovirus messenger RNA: Evidence for a scanning mechanism

A scanning mechanism has been proposed (Kozak, 1978) to explain how eukaryotic ribosomes select the correct AUG codon for initiation of protein synthesis. The hypothesis is that a 40 S ribosomal subunit binds initially at or near the 5′-terminus of a message and subsequently migrates toward the interior of the messenger RNA, stopping when it encounters the first AUG codon, at which point a 60 S subunit joins and peptide bond formation begins. The scanning mechanism predicts that if a message were modified by introduction of a new AUG triplet upstream of the existing initiator codon, the adventitious AUG should be the preferred site for formation of an 80 S initiation complex. This prediction has been confirmed in the present studies with two reovirus messenger RNAs, in which sodium bisulfite was used to convert an ACG sequence (located in the 5′ untranslated region of each message) to AUG. Analysis of the ribosome-protected mRNA fragments recovered from sparsomycin-blocked 80 S initiation complexes revealed that a high percentage of wheat germ ribosomes were centered around the “unnatural” 5′-proximal AUG created by the bisulfite treatment, although some ribosomes were also positioned at the second (normal) initiator codon. The bisulfite modification was carried out in 7 m-urea at 37 °C. resulting in quantitative conversion of cytosine to uracil. Thus, both the primary and secondary structure of the message were drastically altered. These perturbations did not impair the efficiency of ribosome binding, nor did the highly unfolded state of the mRNA permit ribosomes to attach to spurious sites in the interior of the message. The data support a mechanism in which the initiator codon is selected by virtue of its position in a message (i.e. closest to the 5′-terminus), without regard to either the primary or secondary structure of the flanking regions.     

Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli.

Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-1-deoxyribulose 5''-phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones. A strain of E. coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady-state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184.     

Evolutionary Morphing of Tryptophan Synthase: Functional Mechanisms for the Enzymatic Channeling of Indole

Tryptophan synthase (TrpS) is a heterotetrameric αββα enzyme that exhibits complex substrate channeling and allosteric mechanisms and is a model system in enzymology. In this work, we characterize proposed early and late evolutionary states of TrpS and show that they have distinct quaternary structures caused by insertions–deletions of sequence segments (indels) in the β-subunit. Remarkably, indole hydrophobic channels that connect α and β active sites have re-emerged in both TrpS types, yet they follow different paths through the β-subunit fold. Also, both TrpS geometries activate the α-subunit through the rearrangement of loops flanking the active site. Our results link evolutionary sequence changes in the enzyme subunits with channeling and allostery in the TrpS enzymes. The findings demonstrate that indels allow protein quaternary architectures to escape “minima” in the evolutionary landscape, thereby overcoming the conservational constraints imposed by existing functional interfaces and being free to morph into new mechanistic enzymes.     

Temporal resource partitioning mitigates interspecific competition and promotes coexistence among insect parasites

A key to understanding life's great diversity is discerning how competing organisms divide limiting resources to coexist in diverse communities. While temporal resource partitioning has long been hypothesized to reduce the negative effects of interspecific competition, empirical evidence suggests that time may not often be an axis along which animal species routinely subdivide resources. Here, we present evidence to the contrary in the world's most biodiverse group of animals: insect parasites (parasitoids). Specifically, we conducted a meta-analysis of 64 studies from 41 publications to determine if temporal resource partitioning via variation in the timing of a key life-history trait, egg deposition (oviposition), mitigates interspecific competition between species pairs sharing the same insect host. When competing species were manipulated to oviposit at (or near) the same time in or on a single host in the laboratory, competition was common, and one species was typically inherently superior (i.e. survived to adulthood a greater proportion of the time). In most cases, however, the inferior competitor could gain a survivorship advantage by ovipositing earlier (or in a smaller number of cases later) into shared hosts. Moreover, this positive (or in a few cases negative) priority advantage gained by the inferior competitor increased as the interval between oviposition times became greater. The results from manipulative experiments were also correlated with patterns of life-history timing and demography in nature: the more inherently competitively inferior a species was in the laboratory, the greater the interval between oviposition times of taxa in co-occurring populations. Additionally, the larger the interval between oviposition times of competing taxa, the more abundant the inferior species was in populations where competitors were known to coexist. Overall, our findings suggest that temporal resource partitioning via variation in oviposition timing may help to facilitate species coexistence and structures diverse insect communities by altering demographic measures of species success. We argue that the lack of evidence for a more prominent role of temporal resource partitioning in promoting species coexistence may reflect taxonomic differences, with a bias towards larger-sized animals. For smaller species like parasitic insects that are specialized to attack one or a group of closely related hosts, have short adult lifespans and discrete generation times, compete directly for limited resources in small, closed arenas and have life histories constrained by host phenology, temporal resource subdivision via variation in life history may play a critical role in allowing species to coexist by alleviating the negative effects of interspecific competition.