Pseudouridine modification in Caenorhabditis elegans spliceosomal snRNAs: unique modifications are found in regions involved in snRNA-snRNA interactions.
BMC molecular biology. 6:20. Abstract
BACKGROUND: Pseudouridine (Psi) is an abundant modified nucleoside in RNA and a number of studies have shown that the presence of Psi affects RNA structure and function. The positions of Psi in spliceosomal small nuclear RNAs (snRNAs) have been determined for a number of species but not for the snRNAs from Caenorhabditis elegans (C. elegans), a popular experimental model system of development. RESULTS: As a prelude to determining the function of or requirement for this modification in snRNAs, we have mapped the positions of Psi in U1, U2, U4, U5, and U6 snRNAs from worms using a specific primer extension method. As with other species, C. elegans U2 snRNA has the greatest number of Psi residues, with nine, located in the 5' half of the U2 snRNA. U5 snRNA has three Psis, in or near the loop of the large stem-loop that dominates the structure of this RNA. U6 and U1 snRNAs each have one Psi, and two Psi residues were found in U4 snRNA. CONCLUSION: The total number of Psis found in the snRNAs of C. elegans is significantly higher than the minimal amount found in yeasts but it is lower than that seen in sequenced vertebrate snRNAs. When the actual sites of modification on C. elegans snRNAs are compared with other sequenced snRNAs most of the positions correspond to modifications found in other species. However, two of the positions modified on C. elegans snRNAs are unique, one at position 28 on U2 snRNA and one at position 62 on U4 snRNA. Both of these modifications are in regions of these snRNAs that interact with U6 snRNA either in the spliceosome or in the U4/U6 small nuclear ribonucleoprotein particle (snRNP) and the presence of Psi may be involved in strengthening the intermolecular association of the snRNAs.
Caenorhabditis elegans pseudouridine synthase 1 activity in vivo: tRNA is a substrate, but not U2 small nuclear RNA.
The Biochemical journal. 372:595-602. Abstract
The formation of pseudouridine (Psi) from uridine is post-transcriptional and catalysed by pseudouridine synthases, several of which have been characterized from eukaryotes. Pseudouridine synthase 1 (Pus1p) has been well characterized from yeast and mice. In yeast, Pus1p has been shown to have dual substrate specificity, modifying uridines in tRNAs and at position 44 in U2 small nuclear RNA (U2 snRNA). In order to study the in vivo activity of a metazoan Pus1p, a knockout of the gene coding for the homologue of Pus1p in Caenorhabditis elegans was obtained. The deletion encompasses the first two putative exons and includes the essential aspartate that is required for activity in truA pseudouridine synthases. The locations of most modified nucleotides on small RNAs in C. elegans are not known, and the positions of Psi were determined on four tRNAs and U2 snRNA. The uridine at position 27 of tRNA(Val) (AAC), a putative Pus1p-modification site, was converted into Psi in the wild-type worms, but the tRNA(Val) (AAC) from mutant worms lacked the modification. Psi formation at positions 13, 32, 38 and 39, all of which should be modified by other pseudouridine synthases, was not affected by the loss of Pus1p. The absence of Pus1p in C. elegans had no effect on the modification of U2 snRNA in vivo, even though worm U2 snRNA has a Psi at position 45 (the equivalent of yeast U2 snRNA position 44) and at four other positions. This result was unexpected, given the known dual specificity of yeast Pus1p.
Systematic structure-activity analysis of microcin J25..
The Journal of biological chemistry. 283(37):25589-95. Abstract
Microcin J25 (MccJ25) is a 21-residue plasmid-encoded ribosomally synthesized lariat-protoknot antibacterial peptide that targets bacterial RNA polymerase. MccJ25 consists of an 8-residue cycle followed by a 13-residue tail that loops back and threads through the cycle. We have performed systematic mutational scanning of MccJ25, constructing and analyzing more than 380 singly substituted derivatives of MccJ25. The results define residues important for production of MccJ25 (comprising synthesis of MccJ25 precursor, processing of MccJ25 precursor, export of mature MccJ25, and stability of mature MccJ25), inhibition of RNA polymerase, and inhibition of bacterial growth. The results show that only a small number of residues (three in the cycle and one in the threaded segment of the tail) are important for MccJ25 production. The results further show that only a small number of additional residues (two in the cycle and four in the threaded segment of the tail) are important for inhibition of transcription. The results open the way for design and construction of more potent MccJ25-based inhibitors of bacterial growth.
Determination of the orientation of a DNA binding motif in a protein-DNA complex by photocrosslinking..
Proceedings of the National Academy of Sciences of the United States of America. 89(21):10287-91. Abstract
We have developed a straightforward biochemical method to determine the orientation of the DNA binding motif of a sequence-specific DNA binding protein relative to the DNA site in the protein-DNA complex. The method involves incorporation of a photoactivatable crosslinking agent at a single site within the DNA binding motif of the sequence-specific DNA binding protein, formation of the derivatized protein-DNA complex, UV-irradiation of the derivatized protein-DNA complex, and determination of the nucleotide(s) at which crosslinking occurs. We have applied the method to catabolite gene activator protein (CAP). We have constructed and analyzed two derivatives of CAP: one having a phenyl azide photoactivatable crosslinking agent at amino acid 2 of the helix-turn-helix motif of CAP, and one having a phenyl azide photoactivatable crosslinking agent at amino acid 10 of the helix-turn-helix motif of CAP. The results indicate that amino acid 2 of the helix-turn-helix motif is close to the top-strand nucleotides of base pairs 3 and 4 of the DNA half site in the CAP-DNA complex, and that amino acid 10 of the helix-turn-helix motif is close to the bottom-strand nucleotide of base pair 10 of the DNA half site in the CAP-DNA complex. The results define unambiguously the orientation of the helix-turn-helix motif relative to the DNA half site in the CAP-DNA complex. Comparison of the results to the crystallographic structure of the CAP-DNA complex [Schultz, S., Shields, S. & Steitz, T. (1991) Science 253, 1001-1007] indicates that the method provides accurate, high-resolution proximity and orientation information.
High-specificity DNA cleavage agent: design and application to kilobase and megabase DNA substrates..
Science (New York, N.Y.). 265(5174):959-62. Abstract
Strategies to cleave double-stranded DNA at specific DNA sites longer than those of restriction endonucleases (longer than 8 base pairs) have applications in chromosome mapping, chromosome cloning, and chromosome sequencing--provided that the strategies yield high DNA-cleavage efficiency and high DNA-cleavage specificity. In this report, the DNA-cleaving moiety copper:o-phenanthroline was attached to the sequence-specific DNA binding protein catabolite activator protein (CAP) at an amino acid that, because of a difference in DNA bending, is close to DNA in the specific CAP-DNA complex but is not close to DNA in the nonspecific CAP-DNA complex. The resulting CAP derivative, OP26CAP, cleaved kilobase and megabase DNA substrates at a 22-base pair consensus DNA site with high efficiency and exhibited no detectable nonspecific DNA-cleavage activity.
Transcriptional Regulation of the SMK1 Mitogen-activated Protein Kinase gene During Meiotic Development in Saccharomyces Cerevisiae.
Mol Cell Biol. 18:5970-5980. Abstract
Meiotic development (sporulation) in Saccharomyces cerevisiae is characterized by an ordered pattern of gene expression, with sporulation-specific genes classified as early, middle, mid-late, or late depending on when they are expressed. SMK1 encodes a mitogen-activated protein kinase required for spore morphogenesis that is expressed as a middle sporulation-specific gene. Here, we identify the cis-acting DNA elements that regulate SMK1 transcription and characterize the phenotypes of mutants with altered expression patterns. The SMK1 promoter contains an upstream activating sequence (UASS) that specifically interacts with the transcriptional activator Abf1p. The Abf1p-binding sites from the early HOP1 and the middle SMK1 promoters are functionally interchangeable, demonstrating that these elements do not play a direct role in their differential transcriptional timing. Timing of SMK1 expression is determined by another cis-acting DNA sequence termed MSE (for middle sporulation element). The MSE is required not only for activation of SMK1 transcription during middle sporulation but also for its repression during vegetative growth and early meiosis. In addition, the SMK1 MSE can repress vegetative expression in the context of the HOP1 promoter and convert HOP1 from an early to a middle gene. SMK1 function is not contingent on its tight transcriptional regulation as a middle sporulation-specific gene. However, promoter mutants with different quantitative defects in SMK1 transcript levels during middle sporulation show distinct sporulation phenotypes.
Sum1 and Ndt80 Proteins Compete for Binding to Middle Sporulation Element Sequences that Control Meiotic gene Expression.
Mol Cell Biol. 23:4814-4825. Abstract
A key transition in meiosis is the exit from prophase and entry into the nuclear divisions, which in the yeast Saccharomyces cerevisiae depends upon induction of the middle sporulation genes. Ndt80 is the primary transcriptional activator of the middle sporulation genes and binds to a DNA sequence element termed the middle sporulation element (MSE). Sum1 is a transcriptional repressor that binds to MSEs and represses middle sporulation genes during mitosis and early sporulation. We demonstrate that Sum1 and Ndt80 have overlapping yet distinct sequence requirements for binding to and acting at variant MSEs. Whole-genome expression analysis identified a subset of middle sporulation genes that was derepressed in a sum1 mutant. A comparison of the MSEs in the Sum1-repressible promoters and MSEs from other middle sporulation genes revealed that there are distinct classes of MSEs. We show that Sum1 and Ndt80 compete for binding to MSEs and that small changes in the sequence of an MSE can yield large differences in which protein is bound. Our results provide a mechanism for differentially regulating the expression of middle sporulation genes through the competition between the Sum1 repressor and the Ndt80 activator.
Blood lead concentrations in a remote Himalayan population.
Science (New York, NY). 210:1135-7. Abstract
The lead content in the air at the foothills of the Himalayas in Nepal was found to be negligible. The concentration of lead in the blood of 103 children and adults living in this region was found to average 3.4 micrograms per deciliter, a level substantially lower than that found in industrialized populations.
Structural basis for cAMP-mediated allosteric control of the catabolite activator protein..
Proceedings of the National Academy of Sciences of the United States of America. 106(17):6927-32. Abstract
The cAMP-mediated allosteric transition in the catabolite activator protein (CAP; also known as the cAMP receptor protein, CRP) is a textbook example of modulation of DNA-binding activity by small-molecule binding. Here we report the structure of CAP in the absence of cAMP, which, together with structures of CAP in the presence of cAMP, defines atomic details of the cAMP-mediated allosteric transition. The structural changes, and their relationship to cAMP binding and DNA binding, are remarkably clear and simple. Binding of cAMP results in a coil-to-helix transition that extends the coiled-coil dimerization interface of CAP by 3 turns of helix and concomitantly causes rotation, by approximately 60 degrees , and translation, by approximately 7 A, of the DNA-binding domains (DBDs) of CAP, positioning the recognition helices in the DBDs in the correct orientation to interact with DNA. The allosteric transition is stabilized further by expulsion of an aromatic residue from the cAMP-binding pocket upon cAMP binding. The results define the structural mechanisms that underlie allosteric control of this prototypic transcriptional regulatory factor and provide an illustrative example of how effector-mediated structural changes can control the activity of regulatory proteins.
Dynamically driven protein allostery..
Nature structural & molecular biology. 13(9):831-8. Abstract
Allosteric interactions are typically considered to proceed through a series of discrete changes in bonding interactions that alter the protein conformation. Here we show that allostery can be mediated exclusively by transmitted changes in protein motions. We have characterized the negatively cooperative binding of cAMP to the dimeric catabolite activator protein (CAP) at discrete conformational states. Binding of the first cAMP to one subunit of a CAP dimer has no effect on the conformation of the other subunit. The dynamics of the system, however, are modulated in a distinct way by the sequential ligand binding process, with the first cAMP partially enhancing and the second cAMP completely quenching protein motions. As a result, the second cAMP binding incurs a pronounced conformational entropic penalty that is entirely responsible for the observed cooperativity. The results provide strong support for the existence of purely dynamics-driven allostery.