Biomedical subjects

W H McClain

Publications and source records attributed to W H McClain.

At least 19 recordsLinked to original sources

PubMed2002

Isolation of novel tRNA(Ala) mutants by library selection in a tRNA(Ala) knockout strain.

The relationship between tRNA structure and function has been widely investigated by site-directed mutagenesis. This method has been a very useful tool to reveal the critical bases in tRNAs that are important for recognition and aminoacylation, but has been limited by the large number of possible base combinations in tRNA molecules. We have devised a new method that uses tRNA knockout cells for selection of functional tRNAs from a mutant tRNA gene library to overcome this limitation. To explore the mechanism of tRNA(Ala) recognition, the bases of the acceptor-stem region were randomized and active mutants were selected in a tRNA(Ala) knockout strain. Mutants of tRNA(Ala) having diverse sequence combinations in the acceptor-stem region and a broad range of functional activity to support knockout cell growth were isolated. The mutant tRNAs selected by the method included molecules containing novel base substitutions as well as extensively altered base combinations that would not be readily generated by rationally designed site-directed mutagenesis. Our results emphasize the importance of the acceptor stem as a structural unit in which some nucleotides may carry more weight than others, but in summation every nucleotide contributes to the interaction with the enzyme.

Amino Acyl-tRNA Synthetases↗

PubMed2001

The structure of an AspRS-tRNA(Asp) complex reveals a tRNA-dependent control mechanism.

The 2.6 A resolution crystal structure of an inactive complex between yeast tRNA(Asp) and Escherichia coli aspartyl-tRNA synthetase reveals the molecular details of a tRNA-induced mechanism that controls the specificity of the reaction. The dimer is asymmetric, with only one of the two bound tRNAs entering the active site cleft of its subunit. However, the flipping loop, which controls the proper positioning of the amino acid substrate, acts as a lid and prevents the correct positioning of the terminal adenosine. The structure suggests that the acceptor stem regulates the loop movement through sugar phosphate backbone- protein interactions. Solution and cellular studies on mutant tRNAs confirm the crucial role of the tRNA three-dimensional structure versus a specific recognition of bases in the control mechanism.

Aspartate-tRNA Ligase↗

PubMed2001

Construction of an Escherichia coli knockout strain for functional analysis of tRNA(Asp).

The specific aminoacylation of tRNA is critical for translation of the genetic code. A molecular description of aminoacylation requires knowledge of the relevant three-dimensional structures, biochemical parameters and the structure-function relationship of the synthetase and its substrate tRNA. Extensive structural and biochemical data are available on the aspartic acid system of Escherichia coli, but there is a paucity of cellular functional data. We have developed a system to overcome this deficiency by engineering an E. coli knockout tRNA(Asp) strain, thereby allowing a penetrating analysis of tRNA(Asp) structure and function under conditions that prevail in the cell.

Acylation↗

PubMed2001

Plasmid systems to study RNA function in Escherichia coli.

Determining the functional activity of an essential RNA in vivo presents special challenges. We have devised an in vivo analysis of alternative forms of an essential tRNA gene in Escherichia coli knockout cells using either a plasmid switch or a regulated two-plasmid system. The model system is presented together with a description of the new plasmids and procedures necessary to effect these analyses. The system is readily adaptable to non-essential RNAs.

Arabinose↗

PubMed2000

The G x U wobble base pair. A fundamental building block of RNA structure crucial to RNA function in diverse biological systems.

The G x U wobble base pair is a fundamental unit of RNA secondary structure that is present in nearly every class of RNA from organisms of all three phylogenetic domains. It has comparable thermodynamic stability to Watson-Crick base pairs and is nearly isomorphic to them. Therefore, it often substitutes for G x C or A x U base pairs. The G x U wobble base pair also has unique chemical, structural, dynamic and ligand-binding properties, which can only be partially mimicked by Watson-Crick base pairs or other mispairs. These features mark sites containing G x U pairs for recognition by proteins and other RNAs and allow the wobble pair to play essential functional roles in a remarkably wide range of biological processes.

Base Pairing↗

PubMed1999

Correlation of deformability at a tRNA recognition site and aminoacylation specificity.

The fidelity of protein synthesis depends on specific tRNA aminoacylation by aminoacyl-tRNA synthetase enzymes, which in turn depends on the recognition of the identity of particular nucleotides and structural features in the substrate tRNA. These features generally reside within the acceptor helix, the anticodon stem-loop, and in some systems the variable pocket of the tRNA. In the alanine system, fidelity is ensured by a G.U wobble base pair located at the third position within the acceptor helix of alanine tRNA. We have investigated the activity of mutant alanine tRNAs to explore the mechanism of enzyme recognition. Here we show that the mismatched pair C-C is an excellent substitute for G.U in alanine-tRNA-knockout cells. A structural investigation by NMR spectroscopy of the C-C RNA acceptor end reveals that the two cytosines are intercalated into the helix, and that C-C exists in multiple conformations. Structural heterogeneity also is present in the wild-type G.U RNA, whereas inactive Watson-Crick helices are structurally rigid. The correlation between functional and structural data suggests that the G.U pair provides a distinctive structure and a point of deformability that allow the tRNA acceptor end to fit into the active site of the alanyl-tRNA synthetase. Fidelity is ensured because noncognate and inactive mutant tRNAs are bound in the active site in an incorrect conformation that reduces enzymatic activity.

Acylation↗

PubMed1999

The reliability of in vivo structure-function analysis of tRNA aminoacylation.

The G.U wobble base-pair in the acceptor helix of Escherichia coli tRNAAlais critical for aminoacylation by the alanine synthetase. Previous work by several groups probed the mechanism of enzyme recognition of G.U by a structure-function analysis of mutant tRNAs using either a cell assay (amber suppressor tRNA) or a test tube assay (phage T7 tRNA substrate and purified enzyme). However, the aminoacylation capacity of particular mutant tRNAs was about 10(4)-fold higher in the cell assay. This led us to scrutinize the cell assay to determine if any parameter exaggerates the extent of aminoacylation in mutants forming substantial amounts of alanyl-tRNAAla. In doing so, we have refined and developed experimental designs to analyze tRNA function. We examined the level of aminoacylation of amber suppressor tRNAAlawith respect to the method of isolating aminoacyl-tRNA, the rate of cell growth, the cellular levels of alanine synthetase and elongation factor TU (EF-Tu), the amount of tRNA and the characteristics of EF-Tu binding. Within the precision of our measurements, none of these parameters varied in a way that could significantly amplify cellular alanyl-tRNAAla. A key observation is that the extent of aminoacylation of tRNAAlawas independent of tRNAAlaconcentration over a 75-fold range. Therefore, the cellular assay of tRNAAlareflects the substrate quality of the molecule for formation of alanyl-tRNAAla. These experiments support the authenticity of the cellular assay and imply that a condition or factor present in the cell assay may be absent in the test tube assay.

Acylation↗

PubMed1999

A set of plasmids constitutively producing different RNA levels in Escherichia coli.

New plasmids were developed for the in vivo expression of RNA in Escherichia coli. These plasmids combine constitutive promoters of different strengths with different origins of replication to provide a 75-fold range of expression of amber suppressor tRNA. The plasmids are either pMB1, p15A or temperature-sensitive SC101 replicons, and can be used in two plasmid systems for studying RNA-protein interactions. The temperature-sensitive SC101 plasmids may be useful as gene replacement vectors. Another vector that is suitable for generating lethal mutations was constructed in a plasmid containing a regulatable phage T7 promoter.

Escherichia coli↗

PubMed1999

Functional compensation by particular nucleotide substitutions of a critical G*U wobble base-pair during aminoacylation of transfer RNA.

Expression of the genetic code depends on precise tRNA aminoacylation by cognate aminoacyl-tRNA synthetase enzymes. The G.U wobble base-pair in the acceptor helix of Escherichia coli alanine tRNA is the primary aminoacylation determinant of this molecule. Previous work on the process of synthetase recognition of the G.U pair showed that replacing G.U by a G.C Watson-Crick base-pair inactivates alanine acceptance by the tRNA, but that C.A and G.A wobble pair replacements preserve acceptance. Work by another group reported that the effects of a G.C replacement were reversed by a distal wobble base-pair in the anticodon helix. This result is potentially interesting because it suggests that distant regions in alanine tRNA are functionally coupled during synthetase recognition and more generally because recognition determinants of many other tRNAs lie in both the acceptor helix and anticodon helix region. Here, we have conducted an extensive in vivo analysis of the distal wobble pair in alanine tRNA and report that it does not behave like a compensating mutation. Restoration of alanine acceptance was not detected even when the synthetase enzyme was overproduced. We discuss the previous experimental evidence and suggest how the distal wobble pair was incorrectly analyzed. The available data indicate that all principal recognition determinants of alanine tRNA lie in the molecule's acceptor helix.

Acylation↗

PubMed1999

The relationship of thermodynamic stability at a G x U recognition site to tRNA aminoacylation specificity.

The G x U pair at the third position in the acceptor helix of Escherichia coli tRNA(Ala) is critical for aminoacylation. The features that allow G x U recognition are likely to include direct interaction of alanyl-tRNA synthetase with distinctive atomic groups and indirect recognition of the structural and stability information encoded in the sequence of G x U and its immediate context. The present work investigates the thermodynamic stability and acceptor activity for a comprehensive set of variant RNAs with substitutions of the G x U pair of E. coli tRNA(Ala). The four RNAs with Watson-Crick substitutions had a lower acceptor activity and a higher stability relative to the G x U RNA. On the other hand, the RNAs with mispair substitutions had a lower stability, but either a higher or a lower acceptor activity. Thus, the entire set of variant RNAs does not exhibit a correlation between thermodynamic stability of the free, unbound tRNA and its acceptor activity. The substantial acceptor activity of tRNAs with particular mispair substitutions may be explained by their ability to assume the conformational preferences of alanyl-tRNA synthetase. Moreover, the G x U pair may provide a point of deformability for the substrate tRNA to adapt to the enzyme's active site.

Base Pairing↗

PubMed1998

The importance of tRNA backbone-mediated interactions with synthetase for aminoacylation.

We have identified six new aminoacylation determinants of Escherichia coli tRNAGln in a genetic and biochemical analysis of suppressor tRNA. The new determinants occupy the interior of the acceptor stem, the inside corner of the L shape, and the anticodon loop of the molecule. They supplement the primary determinants located in the anticodon and acceptor end of tRNAGln described previously. Remarkably, the three-dimensional structure of the complex between tRNAGln and glutaminyl-tRNA synthetase shows that the enzyme interacts with the phosphate-sugar backbone but not the base of every new determinant. Moreover, a small protein motif interacts with five of these determinants, and it binds proximal to the sixth. The motif also interacts with the middle base of the anticodon and with the backbones of six other nucleotides. Our results emphasize that synthetase recognition of tRNA is more elaborate than amino acid side chains of the enzyme interacting with nucleotide bases of the tRNA. Recognition also includes synthetase interaction with tRNA backbone functionalities whose distinctive locations in three-dimensional space are exquisitely determined by the tRNA sequence.

Acylation↗

PubMed1998

tRNA nucleotide 47: an evolutionary enigma.

A previous analysis of tRNA sequences suggested a correlation between the absence of a nucleotide at position 47 (nt 47) in the extra loop and the presence of a U13:G22 base pair in the D-stem. We have evaluated the significance of this correlation by determining the in vivo activity of tRNAs containing either a C13:G22 or a U13:G22 pair in tRNA molecules with or without nt 47. Although this correlation might reflect some malfunction of tRNAs lacking nt 47, but containing the C13:G22, assays of the in vivo suppressor activity showed that this tRNA is actually more active than the tRNA with the features found in the database, i.e., a U13:G22 base pair and no nt 47. Moreover, analogous constructs with a GGC anticodon permitted the growth of an Escherichia coli strain deleted for tRNA(Ala)GGC genes equally well. On the other hand, long-term growth experiments with competing E. coli strains harboring the tRNA lacking nt 47, either with the C13:G22 or the U13:G22 base pair demonstrated that the U13:G22 tRNA overtook the C13:G22 strain even when the starting proportion of strains favored the C13:G22 strain. Thus, the preference for the U13:G22 tRNA lacking nt 47 in the sequence database is most likely due to factors that come into play during extended growth or latency rather than to the ability of the tRNA to engage in protein synthesis.

Amidohydrolases↗

PubMed1996

Functional evidence for indirect recognition of G.U in tRNA(Ala) by alanyl-tRNA synthetase.

The structural features of the G.U wobble pair in Escherichia coli alanine transfer RNA (tRNA(Ala)) that are associated with aminoacylation by alanyl-tRNA synthetase (AlaRS) were investigated in vivo for wild-type tRNA(Ala) and mutant tRNAs with G.U substitutions. tRNA(Ala) with G.U, C.A, or G.A gave similar amounts of charged tRNA(Ala) and supported viability of E. coli lacking chromosomal tRNA(Ala) genes. tRNA(Ala) with G.C was inactive. Recognition of G.U by AlaRS thus requires more than the functional groups on G.U in a regular helix and may involve detection of a helical distortion.

Alanine-tRNA Ligase↗

PubMed1996

Specific function of a G.U wobble pair from an adjacent helical site in tRNA(Ala) during recognition by alanyl-tRNA synthetase.

G.U wobble pairs are crucial to many examples of RNA-protein recognition. We previously concluded that the G.U wobble pair in the acceptor helix of Escherichia coli alanine tRNA (tRNA(Ala)) is recognized indirectly by alanyl-tRNA synthetase (AlaRS), although direct recognition may play some role. Our conclusion was based on the finding that amber suppressor tRNA Ala with G.U shifted to an adjacent helical site retained substantial but incomplete Ala acceptor function in vivo. Other researchers concluded that only direct recognition is operative. We report here a repeat of our original experiment using tRNA(Lys) instead of tRNA(Ala). We find, as in the original experiment, that a shifted G.U confers Ala acceptor activity. Moreover, the modified tRNA(Lys) was specific for Ala, corroborating our original conclusion and making it more compelling.

Alanine-tRNA Ligase↗

PubMed1995

Searching tRNA sequences for relatedness to aminoacyl-tRNA synthetase families.

tRNA sequences were analyzed for sequence features correlated with known classes of aminoacyl-tRNA synthetase enzymes. The tRNAs were searched for distinguishing nucleotides anywhere in their sequences. The analyses did not find nucleotides predictive of synthetase class membership. We conclude that such nucleotides never existed in tRNA sequences or that they existed and were lost from many of the tRNA sequences during evolution.

Amino Acid Sequence↗

PubMed1994

Distinctive acceptor-end structure and other determinants of Escherichia coli tRNAPro identity.

The previously uncharacterized determinants of the specificity of tRNAPro for aminoacylation (tRNAPro identity) were defined by a computer comparison of all Escherichia coli tRNA sequences and tested by a functional analysis of amber suppressor tRNAs in vivo. We determined the amino acid specificity of tRNA by sequencing a suppressed protein and the aminoacylation efficiency of tRNA by examining the steady-state level of aminoacyl-tRNA. On substituting nucleotides derived from the acceptor end and variable pocket of tRNAPro for the corresponding nucleotides in a tRNAPhe gene, the identity of the resulting tRNA changed substantially but incompletely to that of tRNAPro. The redesigned tRNAPhe was weakly active and aminoacyl-tRNA was not detected. Ethyl methanesulfonate mutagenesis of the redesigned tRNAPhe gene produced a mutant with a wobble pair in place of a base pair in the end of the acceptor-stem helix of the transcribed tRNA. This mutant exhibited both a tRNAPro identity and substantial aminoacyl-tRNA. The results speak for the importance of a distinctive conformation in the acceptor-stem helix of tRNAPro for aminoacylation by the prolyl-tRNA synthetase. The anticodon also contributes to tRNAPro identity but is not necessary in vivo.

Amino Acyl-tRNA Synthetases↗

PubMed1993

Rules that govern tRNA identity in protein synthesis.

The specificity of tRNA in protein synthesis depends not only on its recognition of the codon in the mRNA, but also on its recognition of the correct aminoacyl-tRNA synthetase enzyme. The specificity of tRNA in aminoacylation (tRNA identity) depends on the tRNAs productive interaction with the correct enzyme and non-productive interaction with all other enzymes. Although extensive regions of the tRNA interact with the enzyme, only a small number of nucleotides comprise the major determinants of tRNA identity. They often lie in the same positions (acceptor end and anticodon, and variable pocket less often) in different tRNAs. Therefore, a determinant in a given tRNA simultaneously ensures both productive and non-productive interactions with the respective enzymes. Specificity for the acceptor end of the tRNA is achieved, in part, by the specific amino acid sequence within protein binding pocket domains that are part of all aminoacyl-tRNA synthetases. These domains also bind the other two substrates of the enzyme, amino acid and ATP. Specificity for the anticodon and variable pocket of the tRNA is more idiosyncratic. Irrespective of their location in the tRNA, the determinants either interact directly with the enzyme or give the tRNA a conformation for a complementary fit with the enzyme.

Amino Acyl-tRNA Synthetases↗

PubMed1993

Identity of Escherichia coli tRNA(Cys) determined by nucleotides in three regions of tRNA tertiary structure.

The in vivo aminoacylation specificity of tRNA(Cys) was studied with mutants of opal and amber suppressor tRNAs in Escherichia coli. This specificity depends not only on nucleotides in the acceptor end and anticodon of tRNA(Cys), but also on nucleotides in the dihydrouridine stem and loop and the variable loop that interact, forming the complex core of tRNA tertiary structure.

Amino Acid Sequence↗