Evolutionary tuning impacts the design of bacterial tRNAs for the incorporation of unnatural amino acids by ribosomes.

In order to function on the ribosome with uniform rate and adequate accuracy, each bacterial tRNA has evolved to have a characteristic sequence and set of modifications that compensate for the differing physical properties of its esterified amino acid and its codon-anticodon interaction. The sequence of the T-stem of each tRNA compensates for the differential effect of the esterified amino acid on the binding and release of EF-Tu during decoding. The sequence and modifications in the anticodon loop and core of tRNA impact the codon-anticodon strength and the ability of the tRNA to bend during codon recognition.

In vitro selection of DNAs with an increased propensity to form small circles.

A protocol was devised to select for DNA molecules that efficiently form circles from a library of 126 base pair DNAs containing 90 randomized base pairs. After six rounds of selection, individual molecules from the library showed 20- to 100-fold greater j-factors compared with the starting library, validating the selection protocol. High-throughput sequencing revealed a sinusoidal pattern of enrichment and de-enrichment of A/T dinucleotides in the random region with a 10.

Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation.

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process.

The interface between Escherichia coli elongation factor Tu and aminoacyl-tRNA.

Nineteen of the highly conserved residues of Escherichia coli (E. coli) Elongation factor Tu (EF-Tu) that form the binding interface with aa-tRNA were mutated to alanine to better understand how modifying the thermodynamic properties of EF-Tu-tRNA interaction can affect the decoding properties of the ribosome. Comparison of ΔΔG(o) values for binding EF-Tu to aa-tRNA show that the majority of the interface residues stabilize the ternary complex and their thermodynamic contribution can depend on the tRNA species that is used.

Chemical map of Schizosaccharomyces pombe reveals species-specific features in nucleosome positioning.

Using a recently developed chemical approach, we have generated a genome-wide map of nucleosomes in vivo in Schizosaccharomyces pombe (S. pombe) at base pair resolution. The shorter linker length previously identified in S.

tRNA residues evolved to promote translational accuracy.

The decoding properties of 22 structurally conservative base-pair and base-triple mutations in the anticodon hairpin and tertiary core of Escherichia coli tRNA(Ala)GGC were determined under single turnover conditions using E. coli ribosomes. While all of the mutations were able to efficiently decode the cognate GCC codon, many showed substantial misreading of near-cognate GUC or ACC codons.

Duplex destabilization by four ribosomal DEAD-box proteins.

DEAD-box proteins are believed to participate in the folding of RNA by destabilizing RNA secondary or tertiary structures. Although these proteins bind and hydrolyze ATP, the mechanism by which nucleotide hydrolysis is coupled to helix destabilization may vary among different DEAD-box proteins. To investigate their abilities to disrupt helices and couple ATP hydrolysis to unwinding, we assayed the Saccharomyces cerevisiae ribosomal DEAD-box proteins, Dbp3p, Dbp4p, Rok1p, and Rrp3p utilizing a series of RNA substrates containing a short duplex and either a 5' or 3' single-stranded region.

Histidine 66 in Escherichia coli elongation factor tu selectively stabilizes aminoacyl-tRNAs.

The universally conserved His-66 of elongation factor Tu (EF-Tu) stacks on the side chain of the esterified Phe of Phe-tRNA(Phe). The affinities of eight aminoacyl-tRNAs were differentially destabilized by the introduction of the H66A mutation into Escherichia coli EF-Tu, whereas Ala-tRNA(Ala) and Gly-tRNA(Gly) were unaffected. The H66F and H66W proteins each show a different pattern of binding of 10 different aminoacyl-tRNAs, clearly showing that this position is critical in establishing the specificity of EF-Tu for different esterified amino acids.

Is the sequence-specific binding of aminoacyl-tRNAs by EF-Tu universal among bacteria?

Three base pairs in the T-stem are primarily responsible for the sequence-specific interaction of tRNA with Escherichia coli and Thermus thermophilus EF-Tu. While the amino acids on the surface of EF-Tu that contact aminoacyl-tRNA (aa-tRNA) are highly conserved among bacteria, the T-stem sequences of individual tRNA are variable, making it unclear whether or not this protein-nucleic acid interaction is also sequence specific in other bacteria. We propose and validate a thermodynamic model that predicts the ΔG° of any tRNA to EF-Tu using the sequence of its three T-stem base pairs.

Functional consequences of T-stem mutations in E. coli tRNAThrUGU in vitro and in vivo.

The binding affinities between Escherichia coli EF-Tu and 34 single and double base-pair changes in the T stem of E. coli tRNA(Thr)(UGU) were compared with similar data obtained previously for several aa-tRNAs binding to Thermus thermophilus EF-Tu. With a single exception, the two proteins bound to mutations in three T-stem base pairs in a quantitatively identical manner.

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding.

To better understand why aminoacyl-tRNAs (aa-tRNAs) have evolved to bind bacterial elongation factor Tu (EF-Tu) with uniform affinities, mutant tRNAs with differing affinities for EF-Tu were assayed for decoding on Escherichia coli ribosomes. At saturating EF-Tu concentrations, weaker-binding aa-tRNAs decode their cognate codons similarly to wild-type tRNAs. However, tighter-binding aa-tRNAs show reduced rates of peptide bond formation due to slow release from EF-Tu•GDP.

Enhanced product stability in the hammerhead ribozyme.

The rate of dissociation of P1, the 5' product of hammerhead cleavage, is 100-300-fold slower in full-length hammerheads than in hammerheads that either lack or have disrupting mutations in the loop-loop tertiary interaction. The added stability requires the presence of residue 17 at the 3' terminus of P1 but not the 2', 3' terminal phosphate. Since residue 17 is buried within the catalytic core of the hammerhead in the X-ray structure, we propose that the enhanced P1 stability is a result of the cooperative folding of the hammerhead around this residue.

A dominant negative mutant of the E. coli RNA helicase DbpA blocks assembly of the 50S ribosomal subunit.

Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA.

Understanding the sequence specificity of tRNA binding to elongation factor Tu using tRNA mutagenesis.

Measuring the binding affinities of 42 single-base-pair mutants in the acceptor and T Psi C stems of Saccharomyces cerevisiae tRNA Phe to Thermus thermophilus elongation factor Tu (EF-Tu) revealed that much of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into Escherichia coli tRNA CAG Leu resulted in similar changes in binding affinity. Swapping the three pairs from several E.

A sequence element that tunes Escherichia coli tRNA(Ala)(GGC) to ensure accurate decoding.

Mutating the rare A32-U38 nucleotide pair at the top of the anticodon loop of Escherichia coli tRNA(Ala)(GGC) 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 tRNA(Ala)(GGC), the misreading mutant tRNA(Ala)(GGC) shows rapid GTP hydrolysis and no detectable proofreading on near-cognate codons.

Specificity of the ribosomal A site for aminoacyl-tRNAs.

Although some experiments suggest that the ribosome displays specificity for the identity of the esterified amino acid of its aminoacyl-tRNA substrate, a study measuring dissociation rates of several misacylated tRNAs containing the GAC anticodon from the A site showed little indication for such specificity. In this article, an expanded set of misacylated tRNAs and two 2'-deoxynucleotide-substituted mRNAs are used to demonstrate the presence of a lower threshold in k(off) values for aa-tRNA binding to the A site. When a tRNA binds sufficiently well to reach this threshold, additional stabilizing effects due to the esterified amino acid or changes in tRNA sequence are not observed.

Differential RNA-dependent ATPase activities of four rRNA processing yeast DEAD-box proteins.

S. cerevisiae ribosome biogenesis is a highly ordered and dynamic process that involves over 100 accessory proteins, including 18 DExD/H-box proteins that act at discrete steps in the pathway. Although often termed RNA helicases, the biochemical functions of individual DExD/H-box proteins appear to vary considerably.

Different aa-tRNAs are selected uniformly on the ribosome.

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.

Catalytic diversity of extended hammerhead ribozymes.

Chimeras of the well-characterized minimal hammerhead 16 and nine extended hammerheads derived from natural viroids and satellite RNAs were constructed with the goal of assessing whether their very different peripheral tertiary interactions modulate their catalytic properties. For each chimera, three different assays were used to determine the rate of cleavage and the fraction of full-length hammerhead at equilibrium and thereby deduce the elemental cleavage ( k 2) and ligation ( k -2) rate constants. The nine chimeras were all more active than minimal hammerheads and exhibited a very broad range of catalytic properties, with values of k 2 varying by 750-fold and k -2 by 100-fold.

Hammerhead redux: does the new structure fit the old biochemical data?

The cleavage rates of 78 hammerhead ribozymes containing structurally conservative chemical modifications were collected from the literature and compared to the recently determined crystal structure of the Schistosoma mansoni hammerhead. With only a few exceptions, the biochemical data were consistent with the structure, indicating that the new structure closely resembles the transition state of the reaction. Since all the biochemical data were collected on minimal hammerheads that have a very different structure, the minimal hammerhead must be dynamic and occasionally adopt the quite different extended structure in order to cleave.

[3'-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation.

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.

Minimal and extended hammerheads utilize a similar dynamic reaction mechanism for catalysis.

Analysis of the catalytic activity of identical mutations in the catalytic cores of nHH8, a very active "extended" hammerhead, and HH16, a less active "minimal" hammerhead, reveal that the tertiary Watson-Crick base pair between C3 and G8 seen in the recent structure of the Schistosoma mansoni extended hammerhead can be replaced by other base pairs in both backgrounds. This supports the model that both hammerheads utilize a similar catalytic mechanism but HH16 is slower because it infrequently samples the active conformation. The relative effect of different mutations at positions 3 and 8 also depends on the identity of residue 17 in both nHH8 and HH16.

Mutation of the arginine finger in the active site of Escherichia coli DbpA abolishes ATPase and helicase activity and confers a dominant slow growth phenotype.

Escherichia coli DEAD-box protein A (DbpA) is an ATP-dependent RNA helicase with specificity for 23S ribosomal RNA. Although DbpA has been extensively characterized biochemically, its biological function remains unknown. Previous work has shown that a DbpA deletion strain is viable with little or no effect on growth rate.