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.

CYP71BL9, the missing link in costunolide synthesis of sunflower.

Despite intensive research in recent years, the biosynthetic route to costunolide in sunflower so far remained obscured. Additional P450 sequences from public sunflower transcriptomic database were screened to search for candidate enzymes which are able to introduce the 6α-hydroxy-group required for the esterification with the carboxy group of germacarane A acid, the final step in costunolide formation. CYP71BL9, a new P450 enzyme from sunflower was shown to catalyze this hydroxylation, hence being identified as HaCOS. Phylogentically, HaCOS is closer related to HaG8H than to any other known costunolide synthase in Asteraceae.The enzyme was successfully employed to reconstruct the sunflower biosynthesis of costunolide in transformed tobacco. Contrary, in yeast, only minor amounts of sesquiterpene lactone was produced, while 5-hydroxyfarnesylic acid was formed instead. HaCOS in combination with HaG8H produced 8ß-hydroxycostunolide (eupatolide) in transformed plants, thus indicating that sunflower possesses two independent modes of eupatolide synthesis via HaCOS and via HaES. The lack of HaCOS expression and of costunolide in trichomes suggests that the enzyme triggers the costunolied synthesis of the inner tissues of sunflower and might be linked to growth regulation processes.