Ribosome collisions alter frameshifting at translational reprogramming motifs in bacterial mRNAs.

Translational frameshifting involves the repositioning of ribosomes on their messages into decoding frames that differ from those dictated during initiation. Some messenger RNAs (mRNAs) contain motifs that promote deliberate frameshifting to regulate production of the encoded proteins. The mechanisms of frameshifting have been investigated in many systems, and the resulting models generally involve single ribosomes responding to stimulator sequences in their engaged mRNAs. We discovered that the abundance of ribosomes on messages containing the IS3, dnaX, and prfB frameshift motifs significantly influences the levels of frameshifting. We show that this phenomenon results from ribosome collisions that occur during translational stalling, which can alter frameshifting in both the stalled and trailing ribosomes. Bacteria missing ribosomal protein bL9 are known to exhibit a reduction in reading frame maintenance and to have a strong dependence on elongation factor P (EFP). We discovered that ribosomes lacking bL9 become compacted closer together during collisions and that the E-sites of the stalled ribosomes appear to become blocked, which suggests subsequent transpeptidation in transiently stalled ribosomes may become compromised in the absence of bL9. In addition, we determined that bL9 can suppress frameshifting of its host ribosome, likely by regulating E-site dynamics. These findings provide mechanistic insight into the behavior of colliding ribosomes during translation and suggest naturally occurring frameshift elements may be regulated by the abundance of ribosomes relative to an mRNA pool.

A Solvent-Free Approach for Converting Cellulose Waste into Volatile Organic Compounds with Endophytic Fungi.

Simple sugars produced from a solvent-free mechanocatalytic degradation of cellulose were evaluated for suitability as a growth medium carbon source for fungi that produce volatile organic compounds. An endophytic Hypoxylon sp. (CI-4) known to produce volatiles having potential value as fuels was initially evaluated. The growth was obtained on a medium containing the degraded cellulose as the sole carbon source, and the volatile compounds produced were largely the same as those produced from a conventional dextrose/starch diet. A second Hypoxylon sp. (BS15) was also characterized and shown to be phylogenetically divergent from any other named species. The degraded cellulose medium supported the growth of BS15, and approximately the same quantity of the volatile compounds was produced as from conventional diets. Although the major products from BS15 grown on the degraded cellulose were identical to those from dextrose, the minor products differed. Neither CI-4 or BS15 exhibited growth on cellulose that had not been degraded. The extraction of volatiles from the growth media was achieved using solid-phase extraction in order to reduce the solvent waste and more efficiently retain compounds having low vapor pressures. A comparison to more conventional liquid⁻liquid extraction demonstrated that, for CI-4, both methods gave similar results. The solid-phase extraction of BS15 retained a significantly larger variety of the volatile compounds than did the liquid⁻liquid extraction. These advances position the coupling of solvent-free cellulose conversion and endophyte metabolism as a viable strategy for the production of important hydrocarbons.