Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis.
Gene. 26:101-6. Abstract
The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M13mp10 and M13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.
Characterization of the activating region of Escherichia coli catabolite gene activator protein (CAP). I. Saturation and alanine-scanning mutagenesis..
Journal of molecular biology. 243(4):595-602. Abstract
It has been proposed that the surface loop consisting of amino acid residues 152 to 166 of the catabolite gene activator protein (CAP) of Escherichia coli makes direct protein-protein contact with RNA polymerase at the lac promoter. In this work, we have used targeted saturation mutagenesis of codons 152 to 166 of the gene encoding CAP, followed by a screen, to isolate more than 200 independent mutants of CAP defective in transcription activation but not defective in DNA binding. All isolated single-substitution mutants map to just eight amino acid residues; 156, 157, 158, 159, 160, 162, 163 and 164. We propose that these residues define the full extent of the epitope on CAP for the proposed CAP-RNA polymerase interaction. In addition, we have constructed alanine substitutions at each position from residue 152 to 166 of CAP, and we have analyzed the effects on transcription activation at the lac promoter and on DNA binding. Alanine substitution of Thr158 results in an approximately eightfold specific defect in transcription activation. In contrast, alanine substitution of no other residue tested results in a more than twofold specific defect in transcription activation. We conclude that, for Thr158, side-chain atoms beyond C beta are essential for transcription activation at the lac promoter, and we propose that Thr158 OH7 gamma makes direct contact with RNA polymerase in the ternary complex of lac promoter, CAP and RNA polymerase. We conclude further that for no residue other than Thr158 are side-chain atoms beyond C beta essential for transcription activation at the lac promoter.
Transcription activation at class II CAP-dependent promoters: two interactions between CAP and RNA polymerase..
Cell. 87(6):1123-34. Abstract
At Class II catabolite activator protein (CAP)-dependent promoters, CAP activates transcription from a DNA site overlapping the DNA site for RNA polymerase. We show that transcription activation at Class II CAP-dependent promoters requires not only the previously characterized interaction between an activating region of CAP and the RNA polymerase alpha subunit C-terminal domain, but also an interaction between a second, promoter-class-specific activating region of CAP and the RNA polymerase alpha subunit N-terminal domain. We further show that the two interactions affect different steps in transcription initiation. Transcription activation at Class II CAP-dependent promoters provides a paradigm for understanding how an activator can make multiple interactions with the transcription machinery, each interaction being responsible for a specific mechanistic consequence.
Genetic assays to define and characterize protein-protein interactions involved in gene regulation.
Methods. 47:53-62. Abstract
Transcription can be regulated during initiation, elongation, and termination by an enormous variety of regulatory factors. A critical step in obtaining a mechanistic understanding of regulatory factor function is the determination of whether the regulatory factor exerts its effect through direct contact with the transcription machinery. Here I describe the application of a transcription activation-based bacterial two-hybrid assay that is useful for the identification and genetic dissection of protein-protein interactions involved in gene regulation. I provide examples of how this two-hybrid system can be adapted for the study of "global" regulatory factors, sequence-specific DNA-binding proteins, and interactions that occur between two subunits of RNA polymerase (RNAP). These assays facilitate the isolation and characterization of informative amino acid substitutions within both regulatory factors and RNAP. Furthermore, these assays often enable the study of substitutions in essential domains of RNAP that would be lethal in their natural context.
The sigma 70 subunit of RNA polymerase mediates a promoter-proximal pause at the lac promoter..
Nature structural & molecular biology. 11(6):544-50. Abstract
The sigma(70) subunit of RNA polymerase plays an essential role in transcription initiation. In addition, sigma(70) has a critical regulatory role during transcription elongation at the bacteriophage lambda late promoter, lambda P(R'). At this promoter, sigma(70) mediates a pause in early elongation through contact with a DNA sequence element in the initially transcribed region that resembles a promoter -10 element. Here we provide evidence that sigma(70) also mediates a pause in early elongation at the lac promoter (plac). Like that at lambda P(R'), the pause at plac is facilitated by a sequence element in the initially transcribed region that resembles a promoter -10 element. Using biophysical analysis, we demonstrate that the pause-inducing sequence element at plac stabilizes the interaction between sigma(70) and the remainder of the transcription elongation complex. Bioinformatic analysis suggests that promoter-proximal sigma(70)-dependent pauses may play a role in the regulation of many bacterial promoters.
NanoRNAs: A Class of Small RNAs That Can Prime Transcription Initiation in Bacteria.
J Mol Biol. Abstract
It has been widely assumed that all transcription in cells occur using NTPs only (i.e., de novo). However, it has been known for several decades that both prokaryotic and eukaryotic RNA polymerases can utilize small (2 to approximately 5 nt) RNAs to prime transcription initiation in vitro, raising the possibility that small RNAs might also prime transcription initiation in vivo. A new study by Goldman et al. has now provided the first evidence that priming with so-called "nanoRNAs" (i.e., 2 to approximately 5 nt RNAs) can, in fact, occur in vivo. Furthermore, this study provides evidence that altering the extent of nanoRNA-mediated priming of transcription initiation can profoundly influence global gene expression. In this perspective, we summarize the findings of Goldman et al. and discuss the prospect that nanoRNA-mediated priming of transcription initiation represents an underappreciated aspect of gene expression in vivo.
The interaction between sigma70 and the beta-flap of Escherichia coli RNA polymerase inhibits extension of nascent RNA during early elongation..
Proceedings of the National Academy of Sciences of the United States of America. 102(12):4488-93. Abstract
The sigma-subunit of bacterial RNA polymerase (RNAP) is required for promoter-specific transcription initiation. This function depends on specific intersubunit interactions that occur when sigma associates with the RNAP core enzyme to form RNAP holoenzyme. Among these interactions, that between conserved region 4 of sigma and the flap domain of the RNAP beta-subunit (beta-flap) is critical for recognition of the major class of bacterial promoters. Here, we describe the isolation of amino acid substitutions in region 4 of Escherichia coli sigma(70) that have specific effects on the sigma(70) region 4/beta-flap interaction, either weakening or strengthening it. Using these sigma(70) mutants, we demonstrate that the sigma region 4/beta-flap interaction also can affect events occurring downstream of transcription initiation during early elongation. Specifically, our results provide support for a structure-based proposal that, when bound to the beta-flap, sigma region 4 presents a barrier to the extension of the nascent RNA as it emerges from the RNA exit channel. Our findings support the view that the transition from initiation to elongation involves a staged disruption of sigma-core interactions.
Molecular evolution at the decapentaplegic locus in Drosophila.
Genetics. 145:297-309. Abstract
Using an elaborate set of cis-regulatory sequences, the decapentaplegic (dpp) gene displays a dynamic pattern of gene expression during development. The C-terminal portion of the DPP protein is processed to generate a secreted signaling molecule belonging to the transforming growth factor-beta (TGF-beta) family. This signal, the DPP ligand, is able to influence the developmental fates of responsive cells in a concentration-dependent fashion. Here we examine the sequence level organization of a significant portion of the dpp locus in Drosophila melanogaster and use interspecific comparisons with D. simulans, D. pseudoobscura and D.virilis to explore the molecular evolution of the gene. Our interspecific analysis identified significant selective constraint on both the nucleotide and amino acid sequences. As expected, interspecific comparison of protein coding sequences shows that the C-terminal ligand region is highly conserved. However, the central portion of the protein is also conserved, while the N-terminal third is quite variable. Comparison of noncoding regions reveals significant stretches of nucleotide identity in the 3' untranslated portion of exon 3 and in the intron between exons 2 and 3. An examination of cDNA sequences representing five classes of dpp transcripts indicates that these transcripts encode the same polypeptide.
Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains.
BMC Genomics. 9:621. Abstract
ABSTRACT: BACKGROUND: Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR) and methylation spanning linker libraries (MSLL). These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. RESULTS: A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig), while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%). These two techniques were compared with other gene-enrichment methods, and shown to be complementary. CONCLUSION: MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of epigenetic boundaries are barely understood at this time, MSLL technology flags both approximate boundaries and methylated genes that deserve additional investigation. MSLL and HMPR sequences provide a valuable resource for maize genome annotation, and are a uniquely valuable complement to any plant genome sequencing project. In order to make these results fully accessible to the community, a web display was developed that shows the alignment of MSLL, HMPR, and other gene-rich sequences to the BACs; this display is continually updated with the latest ESTs and BAC sequences.
Static and kinetic site-specific protein-DNA photocrosslinking: analysis of bacterial transcription initiation complexes..
Methods in molecular biology (Clifton, N.J.). 543:403-37. Abstract
Static site-specific protein-DNA photocrosslinking permits identification of protein-DNA interactions within multiprotein-DNA complexes. Kinetic site-specific protein-DNA photocrosslinking - involving rapid-quench-flow mixing and pulsed-laser irradiation - permits elucidation of pathways and kinetics of formation of protein-DNA interactions within multiprotein-DNA complexes. We present detailed protocols for application of static and kinetic site-specific protein-DNA photocrosslinking to bacterial transcription initiation complexes.
Structural organization of the RNA polymerase-promoter open complex..
Cell. 101(6):601-11. Abstract
We have used systematic site-specific protein-DNA photocrosslinking to define interactions between bacterial RNA polymerase (RNAP) and promoter DNA in the catalytically competent RNAP-promoter open complex (RPo). We have mapped more than 100 distinct crosslinks between individual segments of RNAP subunits and individual phosphates of promoter DNA. The results provide a comprehensive description of protein-DNA interactions in RPo, permit construction of a detailed model for the structure of RPo, and permit analysis of effects of a transcriptional activator on the structure of RPo.
Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: recognition of pyrimidine-purine and purine-purine steps..
Journal of molecular biology. 357(1):173-83. Abstract
The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A1A2A3T4G5T6G7A8T9C10T11 -3' ("primary kink"). In previous work, we showed that CAP recognizes the nucleotide immediately 5' to the primary-kink site, T6, through an "indirect-readout" mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of approximately 42 degrees, twist angles of approximately 16 degrees ), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of approximately 20 degrees, twist angles of approximately 17 degrees). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, approximately 46 degrees per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, approximately 28 degrees per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.
Identification of a Drosophila gene encoding xylosylprotein beta4-galactosyltransferase that is essential for the synthesis of glycosaminoglycans and for morphogenesis.
The Journal of biological chemistry. 277:46280-8. Abstract
In mammals, the xylosylprotein beta4-galactosyltransferase termed beta4GalT7 (XgalT-1, EC ) participates in proteoglycan biosynthesis through the transfer of galactose to the xylose that initiates each glycosaminoglycan chain. A Drosophila cDNA homologous to mammalian beta4-galactosyltransferases was identified using a human beta4GalT7 cDNA as a probe in a BLAST analysis of expressed sequence tags. The Drosophila cDNA encodes a type II membrane protein with 322 amino acids and shows 49% identity to human beta4GalT7. Extracts from L cells transfected with the cDNA exhibited marked galactosyltransferase activity specific for a xylopyranoside acceptor. Moreover, transfection with the cloned cDNA restored glycosaminoglycan synthesis in beta4GalT7-deficient Chinese hamster ovary cells. In transfectant lysates the properties of Drosophila and human beta4GalT7 resembled each other, except that Drosophila beta4GalT7 showed a less restricted specificity and was active at a wider range of temperatures. Drosophila beta4GalT7 is expressed throughout development, with higher expression levels in adults. Reduction of Drosophila beta4GalT7 levels using expressed RNA interference (RNAi) in imaginal discs resulted in an abnormal wing and leg morphology similar to that of flies with defective Hedgehog and Decapentaplegic signaling, which are known to depend on intact proteoglycan biosynthesis. Immunohistochemical analysis of tissues confirmed that both heparan sulfate and chondroitin sulfate biosynthesis were impaired. Our results demonstrate that Drosophila beta4GalT7 has the in vitro and in vivo properties predicted for an ortholog of human beta4GalT7 and is essential for normal animal development through its role in proteoglycan biosynthesis.
Combined Analysis of Expression data and Transcription Factor Binding Sites in the Yeast Genome.
BMC Genomics. 5:59-59. Abstract
The analysis of gene expression using DNA microarrays provides genome wide profiles of the genes controlled by the presence or absence of a specific transcription factor. However, the question arises of whether a change in the level of transcription of a specific gene is caused by the transcription factor acting directly at the promoter of the gene or through regulation of other transcription factors working at the promoter.