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. 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.

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. pombe is due to a preponderance of nucleosomes separated by ∼4/5 bp, placing nucleosomes on opposite faces of the DNA. The periodic dinucleotide feature thought to position nucleosomes is equally strong in exons as in introns, demonstrating that nucleosome positioning information can be superimposed on coding information. Unlike the case in Saccharomyces cerevisiae, A/T-rich sequences are enriched in S. pombe nucleosomes, particularly at ±20 bp around the dyad. This difference in nucleosome binding preference gives rise to a major distinction downstream of the transcription start site, where nucleosome phasing is highly predictable by A/T frequency in S. pombe but not in S. cerevisiae, suggesting that the genomes and DNA binding preferences of nucleosomes have coevolved in different species. The poly (dA-dT) tracts affect but do not deplete nucleosomes in S. pombe, and they prefer special rotational positions within the nucleosome, with longer tracts enriched in the 10- to 30-bp region from the dyad. S. pombe does not have a well-defined nucleosome-depleted region immediately upstream of most transcription start sites; instead, the -1 nucleosome is positioned with the expected spacing relative to the +1 nucleosome, and its occupancy is negatively correlated with gene expression. Although there is generally very good agreement between nucleosome maps generated by chemical cleavage and micrococcal nuclease digestion, the chemical map shows consistently higher nucleosome occupancy on DNA with high A/T content.

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. Although all the misreading mutations were present in the sequences of other E. coli tRNAs, they were never found among bacterial tRNA(Ala)GGC sequences. This suggests that the sequences of bacterial tRNA(Ala)GGC have evolved to avoid reading incorrect codons.

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 10.4 base pair period associated with the helicity of DNA. A similar, but more moderate pattern of C/G dinucleotides was offset by precisely half a helical turn. While C/G dinucleotide enrichments were evenly distributed, A/T dinucleotide enrichments displayed a preference to cluster in individual DNA molecules. The most highly enriched 10 base pair sequences in the random region contained adjacent blocks of A/T and C/G trinucleotides present in some, but not all, rapidly cyclizing molecules. The phased dinucleotide enrichments closely match those present in accurately mapped yeast nucleosomes, confirming the importance of DNA bending in nucleosome formation. However, at certain sites the nucleosomal DNAs show dinucleotide enrichments that differ substantially from the cyclization data. These discrepancies can often be correlated with sequence specific contacts that form between histones and DNA.

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. Experiments with a very tight binding mutation of tRNA(Tyr) indicate that interface amino acids distant from the tRNA mutation can contribute to the specificity. For nearly all of the mutations, the values of ΔΔG(o) were identical to those previously determined at the orthologous positions of Thermus thermophilus (T. thermophilus) EF-Tu indicating that the thermodynamic properties of the interface were conserved between distantly related bacteria. Measurement of the rate of GTP hydrolysis on programmed ribosomes revealed that nearly all of the interface mutations were able to function in ribosomal decoding. The only interface mutation with greatly impaired GTPase activity was R223A which is the only one that also forms a direct contact with the ribosome. Finally, the ability of the EF-Tu interface mutants to destabilize the EF-Tu-aa-tRNA interaction on the ribosome after GTP hydrolysis were evaluated by their ability to suppress the hyperstable T1 tRNA(Tyr) variant where EF-Tu release is sufficiently slow to limit the rate of peptide bond formation (kpep) . In general, interface mutations that destabilize EF-Tu binding are also able to stimulate kpep of T1 tRNA(Tyr), suggesting that the thermodynamic properties of the EF-Tu-aa-tRNA interaction on the ribosome are quite similar to those found in the free ternary complex.

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. Thus, the affinities of aa-tRNAs for EF-Tu are constrained to be uniform by their need to bind tightly enough to form the ternary complex but weakly enough to release from EF-Tu during decoding. Consistent with available crystal structures, the identity of the esterified amino acid and three base pairs in the T stem of tRNA combine to define the affinity of each aa-tRNA for EF-Tu, both off and on the ribosome.

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. However, the H66A mutation does not greatly affect the ability of the ternary complex to bind ribosomes, hydrolyze GTP, or form dipeptide, suggesting that this residue does not directly participate in ribosomal decoding. Selective mutation of His-66 may improve the ability of certain unnatural amino acids to be incorporated by the ribosome.

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. However, tRNA(Thr) differs from other tRNAs by also using its rare A52-C62 pair as a negative specificity determinant. Using a plasmid-based tRNA gene replacement strategy, we show that many of the tRNA(Thr)(UGU) T-stem changes are either unable to support growth of E. coli or are less effective than the wild-type sequence. Since the inviable T-stem sequences are often present in other E. coli tRNAs, it appears that T-stem sequences in each tRNA body have evolved to optimize function in a different way. Although mutations of tRNA(Thr) can substantially increase or decrease its affinity to EF-Tu, the observed affinities do not correlate with the growth phenotype of the mutations in any simple way. This may either reflect the different conditions used in the two assays or indicate that the T-stem mutants affect another step in the translation mechanism.

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. Despite dramatic differences in T-stem sequences, the predicted ΔG° values for the majority of tRNA classes are similar in all bacteria and closely match the ΔG° values determined for E. coli tRNAs. Each individual tRNA class has evolved to have a characteristic ΔG° value to EF-Tu, but different T-stem sequences are used to achieve this ΔG° value in different bacteria. Thus, the compensatory relationship between the affinity of the tRNA body and the affinity of the esterified amino acid is universal among bacteria. Additionally, we predict and validate a small number of aa-tRNAs that bind more weakly to EF-Tu than expected and thus are candidates for acting as activated amino acid donors in processes outside of translation.

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. All four proteins unwound a 10 bp helix in vitro in the presence of ATP; however, significant dissociation of longer helices was not observed. While Dbp3p did not require a single-stranded extension to disrupt a helix, the unwinding activities of Dbp4p, Rok1p, and Rrp3p were substantially stimulated by either a 5' or 3' single-stranded extension. Interestingly, these proteins showed a clear length dependency with 3' extensions that was not observed with 5' extensions, suggesting that they bind substrates with a preferred orientation. In the presence of AMPPNP or ADP, all four proteins displayed displacement activity suggesting that nucleotide binding is sufficient to facilitate duplex disruption. Further enhancement of the strand displacement rate in the presence of ATP was observed for only Dbp3p and Rrp3p.

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. However, specificity for different amino acid side chains and the tRNA body is observed when tRNA binding is sufficiently weaker than this threshold. We propose that uniform aa-tRNA binding to the A site may be a consequence of a conformational change in the ribosome, induced by the presence of the appropriate combination of contributions from the anticodon, amino acid and tRNA body.

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. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg(2+) exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.

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. However, since P1 is fully stabilized at >2.5 mM MgCl(2) while hammerhead activity continues to increase with an increase in MgCl(2) concentration, it is clear that the hammerhead structure in the transition state must differ from that of the product complex. The product stabilization assay is used to test our earlier proposal that different tertiary interactions modulate the cleavage rate by differentially stabilizing the core.

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. This synergistic effect can best be explained by transient pairing between residues 3 and 17 and 8 and 13, which stabilize an inactive conformation. Thus, mutants of nHH8 and possibly nHH8 itself are also in dynamic equilibrium with an inactive conformation that may resemble the X-ray structure of a minimal hammerhead. Therefore, both minimal and extended hammerhead structures must be considered to fully understand hammerhead catalysis.

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.

Idiosyncratic tuning of tRNAs to achieve uniform ribosome binding.

The binding of seven tRNA anticodons to their complementary codons on Escherichia coli ribosomes was substantially impaired, as compared with the binding of their natural tRNAs, when they were transplanted into tRNA(2)(Ala). An analysis of chimeras composed of tRNA(2)(Ala) and various amounts of either tRNA(3)(Gly) or tRNA(2)(Arg) indicates that the presence of the parental 32-38 nucleotide pair is sufficient to restore ribosome binding of the transplanted anticodons. Furthermore, mutagenesis of tRNA(2)(Ala) showed that its highly conserved A32-U38 pair serves to weaken ribosome affinity. We propose that this negative binding determinant is used to offset the very tight codon-anticodon interaction of tRNA(2)(Ala). This suggests that each tRNA sequence has coevolved with its anticodon to tune ribosome affinity to a value that is the same for all tRNAs.

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. In attempt to elucidate a phenotype for DbpA, point mutations were made at eleven conserved residues in the ATPase active site, which have exhibited dominant-negative phenotypes in other DExD/H proteins. Biochemical analysis of these DbpA mutants shows the expected decrease in RNA-dependent ATPase activity and helix unwinding activity. Only the least biochemically active mutation, R331A, produces small colony phenotype and a reduced growth rate. This dominant slow growth mutant will be valuable to determine the cellular function of DbpA.

Amino acid specificity in translation.

Recent structural and biochemical experiments indicate that bacterial elongation factor Tu and the ribosomal A-site show specificity for both the amino acid and the tRNA portions of their aminoacyl-tRNA (aa-tRNA) substrates. These data are inconsistent with the traditional view that tRNAs are generic adaptors in translation. We hypothesize that each tRNA sequence has co-evolved with its cognate amino acid, such that all aa-tRNAs are translated uniformly.

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. Four DExD/H-box proteins, Dbp3p, Dbp4p, Rok1p, and Rrp3p, involved in yeast ribosome assembly were expressed in E. coli, and all were found to be active RNA-dependent ATPases with k(cat) values ranging from 13 to 170 min(-1) and K(M)(ATP) values ranging from 0.24 to 2.3 mM. All four proteins are activated by single-stranded oligonucleotides, but they require different chain lengths for maximal ATPase activity, ranging from 10 to >40 residues. None of the four proteins shows significant specificity for yeast rRNA, compared to nonspecific control RNAs since these large RNAs contain multiple binding sites that appear to be catalytically similar. This systematic comparison of four members of the DExD/H-box family demonstrates a range of biochemical properties and lays the foundation for relating the activities of proteins to their biological functions.