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Thomas J Santangelo

Publications and source records attributed to Thomas J Santangelo.

12 recordsLinked to original sources
PubMed2026

Phosphate backbone epitranscriptomics: Discovery of natural RNA phosphorothioates and their writer machinery.

Over 150 modifications expand the RNA alphabet, yet all known natural modifications occur on nucleobases or ribose sugars, with none identified on the phosphate backbone. In contrast, phosphorothioates (PSs), in which a non-bridging phosphate oxygen is replaced with sulfur, are central to RNA therapeutics but have never been reliably detected in natural RNAs. Here, we develop sequencing- and mass spectrometry-based approaches to quantitatively map RNA PSs at single-nucleotide resolution. Across diverse archaeal species, we identify stereospecific PS modifications at rRNA and tRNA hotspots, which are dynamically regulated by sulfur availability and temperature. We uncover a diverse enzyme family that selectively modifies tRNA/rRNA substrates and whose evolutionary presence/absence matches the distribution of PSs. Enzyme loss causes inviability or temperature sensitivity, and functional analyses reveal that tRNA PSs enhance tRNA stability. These findings establish the first natural RNA phosphate-backbone modification and its enzymatic machinery, providing a foundation for mechanistic and functional exploration.

RNA modifications↗
PubMed2006

Archaeal minichromosome maintenance (MCM) helicase can unwind DNA bound by archaeal histones and transcription factors.

Protein-DNA complexes must be disassembled to facilitate DNA replication. Replication forks contain a helicase that unwinds the duplex DNA at the front of the fork. The minichromosome maintenance helicase from the archaeon Methanothermobacter thermautotrophicus required only ATP to unwind DNA bound into complexes by the M. thermautotrophicus archaeal histone HMtA2, transcription repressor TrpY, or into a transcription pre-initiation complex by M. thermautotrophicus TATA-box-binding protein, transcription factor B, and RNA polymerase. In contrast, the minichromosome maintenance helicase was unable to unwind DNA bound by this archaeal RNA polymerase in a stalled transcript-elongating complex.

Adenosine Triphosphate↗
PubMed2006

Kinetic investigation of Escherichia coli RNA polymerase mutants that influence nucleotide discrimination and transcription fidelity.

Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site, and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several Escherichia coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue betaAsp(675), which is >10A away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues betaArg(678) and betaAsp(814) do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the betaAsp(675) mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site.

Amino Acid Sequence↗
PubMed2006

Single-molecule analysis of RNA polymerase transcription.

The kinetics and mechanisms of transcription are now being investigated by a repertoire of single-molecule techniques, including optical and magnetic tweezers, high-sensitivity fluorescence techniques, and atomic force microscopy. Single-molecule techniques complement traditional biochemical and crystallographic approaches, are capable of detecting the motions and dynamics of individual RNAP molecules and transcription complexes in real time, and make it possible to directly measure RNAP binding to and unwinding of template DNA, as well as RNAP translocation along the DNA during transcript synthesis.

DNA↗
PubMed2005

Archaeal RNA polymerase is sensitive to intrinsic termination directed by transcribed and remote sequences.

Archaea are prokaryotes with a single DNA-dependent RNA polymerase (RNAP) that is homologous to, and likely resembles the ancestor of all three eukaryotic RNAPs. In vitro studies have confirmed that initiation by archaeal RNAPs resembles the Pol II system, and we report the first detailed in vitro investigation of archaeal transcription termination. Methanothermobacter thermautotrophicus (M.t.) RNAP is susceptible to intrinsic termination at an intergenic sequence that conforms to a bacterial intrinsic terminator, as well as at bona fide bacterial intrinsic terminators. In contrast to bacterial RNAPs, M.t. RNAP also terminated in response to synthetic and natural oligo-T-rich sequences that were not preceded by sequences with any recognizable potential to form a stable RNA hairpin. Both template topology and temperature influenced the position and extent of termination in vitro, and the results argue that transcription of an upstream sequence can alter the termination response of the archaeal RNAP at a remote downstream sequence.

Archaeal Proteins↗
PubMed2005

Cyclic AMP receptor protein and RhaR synergistically activate transcription from the L-rhamnose-responsive rhaSR promoter in Escherichia coli.

The Escherichia coli rhaSR operon encodes two AraC family transcription activator proteins, RhaS and RhaR, which regulate expression of the l-rhamnose catabolic regulon in response to l-rhamnose availability. RhaR positively regulates rhaSR in response to l-rhamnose, and RhaR activation can be enhanced by the cyclic AMP (cAMP) receptor protein (CRP) protein. CRP is a well-studied global transcription regulator that binds to DNA as a dimer and activates transcription in the presence of cAMP. We investigated the mechanism of CRP activation at rhaSR both alone and in combination with RhaR in vivo and in vitro. Base pair substitutions at potential CRP binding sites in the rhaSR-rhaBAD intergenic region demonstrate that CRP site 3, centered at position -111.5 relative to the rhaSR transcription start site, is required for the majority of the CRP-dependent activation of rhaSR. DNase I footprinting confirms that CRP binds to site 3; CRP binding to the other potential CRP sites at rhaSR was not detected. We show that, at least in vitro, CRP is capable of both RhaR-dependent and RhaR-independent activation of rhaSR from a total of three transcription start sites. In vitro transcription assays indicate that the carboxy-terminal domain of the alpha subunit (alpha-CTD) of RNA polymerase is at least partially dispensable for RhaR-dependent activation but that the alpha-CTD is required for CRP activation of rhaSR. Although CRP requires the presence of RhaR for efficient in vivo activation of rhaSR, DNase I footprinting assays indicated that cooperative binding between RhaR and CRP does not make a significant contribution to the mechanism of CRP activation at rhaSR. It therefore appears that CRP activates transcription from rhaSR as it would at simple class I promoters, albeit from a relatively distant position.

Base Sequence↗
PubMed2004

A single-molecule technique to study sequence-dependent transcription pausing.

We present a technique that allows sequence-dependent analysis of transcription elongation using single-molecule optical trapping techniques. Observation of individual molecules of RNA polymerase (RNAP) allows determination of elongation kinetics that are difficult or impossible to accurately obtain from bulk studies, and provides high temporal resolution of the RNAP motion under a calibrated mechanical load. One limitation of previous single molecule studies was the difficulty in correlating the observed motion of RNAP with its actual position on the DNA template to better than approximately 100 bp. In this work, we improved the spatial precision of optical trapping studies of transcription to approximately 5 bp by using runoff transcription as an unambiguous marker of RNAP template position. This runoff method was sufficient to unequivocally locate and study a single known pause sequence (DeltatR2). By applying various loads to assist RNAP forward translocation, we specifically investigated elongation kinetics within this pause region and found that the dwell time at the pause sequence decreased with increasing assisting load. This observation is consistent with bulk biochemical studies that suggest RNAP reverse translocates, or "backtracks," at the DeltatR2 pause sequence.

Base Sequence↗
PubMed2004

Forward translocation is the natural pathway of RNA release at an intrinsic terminator.

Intrinsic terminators of bacterial RNA polymerase are small (< approximately 30 bp) sequences containing a dyad symmetry that encodes a hairpin in the RNA, followed immediately by a uridine-rich stretch of 5-9 nucleotides just before the site of RNA release. Formation of the RNA hairpin destabilizes the elongation complex, leading to transcript release. We test a model in which hair-pin formation drives RNA polymerase and the melted DNA bubble downstream without transcript elongation, thus releasing the transcript from its enclosure within the enzyme as an RNA/DNA hybrid. We show that blocking downstream translocation of RNAP and preventing downstream DNA unwinding both inhibit transcript release. We argue that translocation of RNA polymerase is essential and that translocation of the bubble stimulates, but is not required, for RNA release; we conclude that forward translocation is the natural pathway of RNA release at an intrinsic terminator.

Bacterial Proteins↗
PubMed2003

RNA polymerase mutations that impair conversion to a termination-resistant complex by Q antiterminator proteins.

Bacteriophage lambda Q-protein stably binds and modifies RNA polymerase (RNAP) to a termination-resistant form. We describe amino acid substitutions in RNAP that disrupt Q-mediated antitermination in vivo and in vitro. The positions of these substitutions in the modeled RNAP/DNA/RNA ternary elongation complex, and their biochemical properties, suggest that they do not define a binding site for Q in RNAP, but instead act by impairing interactions among core RNAP subunits and nucleic acids that are essential for Q modification. A specific conjecture is that Q modification stabilizes interactions of RNAP with the DNA/RNA hybrid and optimizes alignment of the nucleic acids in the catalytic site. Such changes would inhibit the activity of the RNA hairpin of an intrinsic terminator to disrupt the 5'-terminal bases of the hybrid and remove the RNA 3' terminus from the active site.

Bacterial Proteins↗
PubMed2002

Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior.

By using single-molecule measurements, we demonstrate that the elongation kinetics of individual Escherichia coli RNA polymerase molecules are remarkably homogeneous. We find no evidence of distinct elongation states among RNA polymerases. Instead, the observed heterogeneity in transcription rates results from statistical variation in the frequency and duration of pausing. When transcribing a gene without strong pause sites, RNA polymerase molecules display transient pauses that are distributed randomly in both time and distance. Transitions between the active elongation mode and the paused state are instantaneous within the resolution of our measurements (<1 s). This elongation behavior is compared with that of a mutant RNA polymerase that pauses more frequently and elongates more slowly than wild type.

DNA-Directed RNA Polymerases↗
PubMed2002

RfaH, a bacterial transcription antiterminator.

The bacterial transcription antiterminator RfaH has been shown to act, in a purified biochemical system, by binding both RNA polymerase and the nontemplate strand of DNA at the regulatory site ops.

DNA, Bacterial↗