Ten E. coli aminoacyl-tRNAs (aa-tRNAs) were assessed for their ability to decode cognate codons on E. coli ribosomes by using three assays that evaluate the key steps in the decoding pathway. Despite a wide variety of structural features, each aa-tRNA exhibited similar kinetic and thermodynamic properties in each assay. This surprising kinetic and thermodynamic uniformity is likely to reflect the importance of ribosome conformational changes in defining the rates and affinities of the decoding process as well as the evolutionary “tuning” of each aa-tRNA sequence to modify their individual interactions with the ribosome at each step.

The analysis of reactions involving amino acids esterified to tRNAs traditionally uses radiolabeled amino acids. We describe here an alternative assay involving [3'-32P]-labeled tRNA followed by nuclease digestion and TLC analysis that permits aminoacylation to be monitored in an efficient, quantitative manner while circumventing many of the problems faced when using radiolabeled amino acids. We also describe a similar assay using [3'-32P]-labeled aa-tRNAs to determine the rate of peptide bond formation on the ribosome. This type of assay can also potentially be adapted to study other reactions involving an amino acid or peptide esterified to tRNA.

Mutating the rare A32-U38 nucleotide pair at the top of the anticodon loop of E. coli tRNAGGCAla to a more common U32-A38 pair results in a tRNA that performs almost normally on cognate codons but is unusually efficient in reading near-cognate codons. Pre-steady state kinetic measurements on E. coli ribosomes show that unlike the wild-type tRNAGGCAla, the misreading mutant tRNAGGCAla shows rapid GTP hydrolysis and no detectable proofreading on near-cognate codons. Similarly, tRNAGGCAla mutated to contain C32-G38, a pair which is found in some bacterial tRNAGGCAla sequences, was able to decode only the cognate codons, while tRNAGGCAla containing a more common C32-A38 pair was able to decode all cognate and near-cognate codons tested. We propose that many of the phylogenetically conserved sequence elements present in each tRNA have evolved to suppress translation of near-cognate codons.

It is commonly assumed that all DEAD-box ATPases function via a shared mechanism, since this is the case for the few proteins characterized thus far. Hodge and colleagues (pp. 1052-1064) and colleagues (pp. 1065-1077) now describe a novel model for Dbp5's ATPase cycle in mRNA (messenger RNA)/protein complex (mRNP) remodeling during nuclear export. Notably, unlike other DEAD-box proteins, Dbp5 uses a conformational change distinct from ATP hydrolysis for its activity and requires an ADP release factor to reset its ATPase cycle. Dbp5 is a canonical DEAD-box protein with a novel mechanismDbp5 is one of 25 DEAD-box RNA-dependent ATPases in budding yeast. One or more DEAD-box proteins are associated with every major step of RNA processing (for recent reviews, see Cordin et al. 2006;Jarmoskaite and Russell 2010). This family of proteins is characterized by nine conserved motifs (including the eponymous DEAD motif), which contact ATP and RNA (Fig. 1A). The conserved motifs fold into two RecA-like domains connected by a flexible linker (Fig. 1B). These domains are loosely associated when nucleotide-free, but form a binding pocket for ATP and a binding surface for RNA that are each contacted by conserved residues within the characteristic motifs. Although only a handful of structures exist for DEAD-box proteins, the RecA-like domains in different proteins interact with nucleotides and RNA very similarly (Caruthers et al. 2000;Cheng et al. 2005;Andersen et al. 2006;Sengoku et al. 2006;. DEAD-box proteins accomplish a wide range of cellular tasks, including remodeling or stabilizing structured RNA, unwinding short helical regions of RNA, stabilizing protein complexes, and directly removing proteins bound to RNA. The functional specificity of individual proteins in the DEAD-box family is due at least in part to their unique N-terminal and C-terminal sequences flanking the two RecA-like domains and their interactions with regulatory protein cofactors.Several DEAD-box proteins from various organisms have been well characterized, and all function via a similar mechanism (Hilbert et al. 2009). The DEAD-box ATPase cycle begins in the absence of nucleotides and RNA with the two RecA-like domains in an open conformation. Next, ATP binding and RNA binding occur in a cooperative manner. When bound to ATP, DEAD-box proteins have a tight affinity for RNA. ATP binding is sufficient for many DEAD-box proteins to unwind short duplex RNA. Structures of RNA-bound DEAD-box proteins show a sharp kink in the backbone of the ssRNA that is incompatible with A-form helical RNA. ATP hydrolysis is not necessary for the unwinding activity of several DEAD-box proteins, which is still possible in the presence of certain nonhydrolyzable ATP analogs. However, full activity seems to require ATP hydrolysis itself, but not release of the inorganic phosphate (P i ) from the hydrolyzed ATP. Once P i is released, the ADP-bound DEAD-box protein has a low affinity for RNA and dissociates from it. Finally, ADP dissociates from the DEAD-box protein, which is...

Pre-mRNA splicing is an essential step of eukaryotic gene expression that requires both high efficiency and high fidelity. Prp8 has long been considered the "master regulator" of the spliceosome, the molecular machine that executes pre-mRNA splicing. Cross-linking and structural studies place the RNaseH domain (RH) of Prp8 near the spliceosome's catalytic core and demonstrate that alleles that map to a 17-aa extension in RH stabilize it in one of two mutually exclusive structures, the biological relevance of which are unknown. We performed an extensive characterization of alleles that map to this extension and, using in vitro and in vivo reporter assays, show they fall into two functional classes associated with the two structures: those that promote error-prone/efficient splicing and those that promote hyperaccurate/inefficient splicing. Identification of global locations of endogenous splice-site activation by lariat sequencing confirms the fidelity effects seen in our reporter assays. Furthermore, we show that error-prone/efficient RH alleles suppress a mutant deficient at promoting the first catalytic step of splicing, whereas hyperaccurate/inefficient RH alleles exhibit synthetic sickness. Together our data indicate that RH alleles link splicing fidelity with catalytic efficiency by biasing the relative stabilities of distinct spliceosome conformations. We hypothesize that the spliceosome "toggles" between such error-prone/efficient and hyperaccurate/inefficient conformations during the splicing cycle to regulate splicing fidelity.