Twin hydroxymethyluracil-A base pair steps define the binding site for the DNA-bending protein TF1

The DNA-bending protein TF1 is the Bacillus subtilis bacteriophage SPO1- encoded homolog of the bacterial HU proteins and the Escherichia coli integration host factor. We recently proposed that TF1, which binds with high affinity (K(d) was ~3 nM) to preferred sites within the hydroxymethyluracil (hmU)-containing phage genome, identifies its binding sites based on sequence-dependent DNA flexibility. Here, we show that two hmU-A base pair steps coinciding with two previously proposed sites of DNA distortion are critical for complex formation. The affinity of TF1 is reduced 10-fold when both of these hmU-A base pair steps are replaced with A-hmU, G-C, or C-G steps; only modest changes in affinity result when substitutions are made at other base pairs of the TF1 binding site. Replacement of all hmU residues with thymine decreases the affinity of TF1 greatly; remarkably, the high affinity is restored when the two hmU-A base pair steps corresponding to previously suggested sites of distortion are reintroduced into otherwise T- containing DNA. T-DNA constructs with 3-base bulges spaced apart by 9 base pairs of duplex also generate nM affinity of TF1. We suggest that twin hmU-A base pair steps located at the proposed sites of distortion are key to target site selection by TF1 and that recognition is based largely, if not entirely, on sequence-dependent DNA flexibility

The Response of Hemostatic Marker Levels to Activated Factor VII in a Neonate following Cardiopulmonary Bypass

The primary function of recombinant activated factor VII (rFVIIa) is to increase thrombin formation which leads to increased fibrin and less “bleeding.” As a result, most of literature utilizes “bleeding” as the outcome measure with respect to rFVIIa. However, we report the actual effect of rFVIIa on changes in hemostatic markers such as prothrombin activation peptide F1.2, thrombin antithrombin complex (TAT), D-dimer, tissue plasminogen activator (tPA), and plasminogen activator inhibitor (PAI) in a neonate after cardiopulmonary bypass. A single dose of rFVIIa caused a 5.5-fold increase in F1.2, 3.5-fold increase in TAT, and a small increase in d-dimer compared to only a 1.5-fold increase, no increase, and a decrease, respectively, in two neonates undergoing the same procedure having not received rFVIIa. The patterns of change for tPA and PAI were similar

A polymerase III-like reinitiation mechanism is operating in regulation of histone expression in archaea

An archaeal histone gene from the hyperthermophile Pyrococcus furiosus containing four consecutive putative oligo-dT terminator sequences was used as a model system to investigate termination signals and the mechanism of termination in vitro. The archaeal RNA polymerase terminated with high efficiency at the first terminator at 90°C when it contained five to six T residues, at 80°C readthrough was significantly increased. A putative hairpin structure upstream of the first terminator had no effect on termination efficiency. Template competition experiments starting with RNA polymerase molecules engaged in ternary complexes revealed recycling of RNA polymerase from the terminator to the promoter of the same template. This facilitated reinitiation was dependent upon the presence of a terminator sequence suggest-ing that pausing at the terminator is required for recycling as in the RNA polymerase III system. Replacement of the sequences immediately down-stream of the oligo-dT terminator by an AT-rich segment improved termination efficiency. Both AT-rich and GC-rich downstream sequences seemed to impair the facilitated reinitiation pathway. Our data suggest that recycling is dependent on a subtle interplay of pausing of RNA polymerase at the ter-minator and RNA polymerase translocation beyond the oligo-dT termination signal that is dramatically affected by downstream sequences

Different sequence signatures in the upstream regions of plant and animal tRNA genes shape distinct modes of regulation

In eukaryotes, the transcription of tRNA genes is initiated by the concerted action of transcription factors IIIC (TFIIIC) and IIIB (TFIIIB) which direct the recruitment of polymerase III. While TFIIIC recognizes highly conserved, intragenic promoter elements, TFIIIB binds to the non-coding 5′-upstream regions of the tRNA genes. Using a systematic bioinformatic analysis of 11 multicellular eukaryotic genomes we identified a highly conserved TATA motif followed by a CAA-motif in the tRNA upstream regions of all plant genomes. Strikingly, the 5′-flanking tRNA regions of the animal genomes are highly heterogeneous and lack a common conserved sequence signature. Interestingly, in the animal genomes the tRNA species that read the same codon share conserved motifs in their upstream regions. Deep-sequencing analysis of 16 human tissues revealed multiple splicing variants of two of the TFIIIB subunits, Bdp1 and Brf1, with tissue-specific expression patterns. These multiple forms most likely modulate the TFIIIB–DNA interactions and explain the lack of a uniform signature motif in the tRNA upstream regions of animal genomes. The anticodon-dependent 5′-flanking motifs provide a possible mechanism for independent regulation of the tRNA transcription in various human tissues

Mutation of RNA Pol III Subunit rpc2/polr3b Leads to Deficiency of Subunit Rpc11 and Disrupts Zebrafish Digestive Development

The role of RNA polymerase III (Pol III) in developing vertebrates has not been examined. Here, we identify a causative mutation of the second largest Pol III subunit, polr3b, that disrupts digestive organ development in zebrafish slim jim (slj) mutants. The slj mutation is a splice-site substitution that causes deletion of a conserved tract of 41 amino acids in the Polr3b protein. Structural considerations predict that the slj Pol3rb deletion might impair its interaction with Polr3k, the ortholog of an essential yeast Pol III subunit, Rpc11, which promotes RNA cleavage and Pol III recycling. We engineered Schizosaccharomyces pombe to carry an Rpc2 deletion comparable to the slj mutation and found that the Pol III recovered from this rpc2-Δ yeast had markedly reduced levels of Rpc11p. Remarkably, overexpression of cDNA encoding the zebrafish rpc11 ortholog, polr3k, rescued the exocrine defects in slj mutants, indicating that the slj phenotype is due to deficiency of Rpc11. These data show that functional interactions between Pol III subunits have been conserved during eukaryotic evolution and support the utility of zebrafish as a model vertebrate for analysis of Pol III function

Maf1, a New Player in the Regulation of Human RNA Polymerase III Transcription

BACKGROUND: Human RNA polymerase III (pol III) transcription is regulated by several factors, including the tumor suppressors P53 and Rb, and the proto-oncogene c-Myc. In yeast, which lacks these proteins, a central regulator of pol III transcription, called Maf1, has been described. Maf1 is required for repression of pol III transcription in response to several signal transduction pathways and is broadly conserved in eukaryotes. METHODOLOGY/PRINCIPAL FINDINGS: We show that human endogenous Maf1 can be co-immunoprecipitated with pol III and associates in vitro with two pol III subunits, the largest subunit RPC1 and the α-like subunit RPAC2. Maf1 represses pol III transcription in vitro and in vivo and is required for maximal pol III repression after exposure to MMS or rapamycin, treatments that both lead to Maf1 dephosphorylation. CONCLUSIONS/SIGNIFICANCE: These data suggest that Maf1 is a major regulator of pol III transcription in human cells

Tissue-Specific Differences in Human Transfer RNA Expression

Over 450 transfer RNA (tRNA) genes have been annotated in the human genome. Reliable quantitation of tRNA levels in human samples using microarray methods presents a technical challenge. We have developed a microarray method to quantify tRNAs based on a fluorescent dye-labeling technique. The first-generation tRNA microarray consists of 42 probes for nuclear encoded tRNAs and 21 probes for mitochondrial encoded tRNAs. These probes cover tRNAs for all 20 amino acids and 11 isoacceptor families. Using this array, we report that the amounts of tRNA within the total cellular RNA vary widely among eight different human tissues. The brain expresses higher overall levels of nuclear encoded tRNAs than every tissue examined but one and higher levels of mitochondrial encoded tRNAs than every tissue examined. We found tissue-specific differences in the expression of individual tRNA species, and tRNAs decoding amino acids with similar chemical properties exhibited coordinated expression in distinct tissue types. Relative tRNA abundance exhibits a statistically significant correlation to the codon usage of a collection of highly expressed, tissue-specific genes in a subset of tissues or tRNA isoacceptors. Our findings demonstrate the existence of tissue-specific expression of tRNA species that strongly implicates a role for tRNA heterogeneity in regulating translation and possibly additional processes in vertebrate organisms

Diversity of tRNA genes in eukaryotes

We compare the diversity of chromosomal-encoded transfer RNA (tRNA) genes from 11 eukaryotes as identified by tRNAScan-SE of their respective genomes. They include the budding and fission yeast, worm, fruit fly, fugu, chicken, dog, rat, mouse, chimp and human. The number of tRNA genes are between 170 and 570 and the number of tRNA isoacceptors range from 41 to 55. Unexpectedly, the number of tRNA genes having the same anticodon but different sequences elsewhere in the tRNA body (defined here as tRNA isodecoder genes) varies significantly (10–246). tRNA isodecoder genes allow up to 274 different tRNA species to be produced from 446 genes in humans, but only up to 51 from 275 genes in the budding yeast. The fraction of tRNA isodecoder genes among all tRNA genes increases across the phylogenetic spectrum. A large number of sequence differences in human tRNA isodecoder genes occurs in the internal promoter regions for RNA polymerase III. We also describe a systematic, ligation-based method to detect and quantify tRNA isodecoder molecules in human samples, and show differential expression of three tRNA isodecoders in six human tissues. The large number of tRNA isodecoder genes in eukaryotes suggests that tRNA function may be more diverse than previously appreciated

A workflow for genome-wide mapping of archaeal transcription factors with ChIP-seq

Deciphering the structure of gene regulatory networks across the tree of life remains one of the major challenges in postgenomic biology. We present a novel ChIP-seq workflow for the archaea using the model organism Halobacterium salinarum sp. NRC-1 and demonstrate its application for mapping the genome-wide binding sites of natively expressed transcription factors. This end-to-end pipeline is the first protocol for ChIP-seq in archaea, with methods and tools for each stage from gene tagging to data analysis and biological discovery. Genome-wide binding sites for transcription factors with many binding sites (TfbD) are identified with sensitivity, while retaining specificity in the identification the smaller regulons (bacteriorhodopsin-activator protein). Chromosomal tagging of target proteins with a compact epitope facilitates a standardized and cost-effective workflow that is compatible with high-throughput immunoprecipitation of natively expressed transcription factors. The Pique package, an open-source bioinformatics method, is presented for identification of binding events. Relative to ChIP-Chip and qPCR, this workflow offers a robust catalog of protein–DNA binding events with improved spatial resolution and significantly decreased cost. While this study focuses on the application of ChIP-seq in H. salinarum sp. NRC-1, our workflow can also be adapted for use in other archaea and bacteria with basic genetic tools

Transcriptionally active TFIIH of the early-diverged eukaryote Trypanosoma brucei harbors two novel core subunits but not a cyclin-activating kinase complex

Trypanosoma brucei is a member of the early-diverged, protistan family Trypanosomatidae and a lethal parasite causing African Sleeping Sickness in humans. Recent studies revealed that T. brucei harbors extremely divergent orthologues of the general transcription factors TBP, TFIIA, TFIIB and TFIIH and showed that these factors are essential for initiating RNA polymerase II-mediated synthesis of spliced leader (SL) RNA, a trans splicing substrate and key molecule in trypanosome mRNA maturation. In yeast and metazoans, TFIIH is composed of a core of seven conserved subunits and the ternary cyclin-activating kinase (CAK) complex. Conversely, only four TFIIH subunits have been identified in T. brucei. Here, we characterize the first protistan TFIIH which was purified in its transcriptionally active form from T. brucei extracts. The complex consisted of all seven core subunits but lacked the CAK sub-complex; instead it contained two trypanosomatid-specific subunits, which were indispensable for parasite viability and SL RNA gene transcription. These findings were corroborated by comparing the molecular structures of trypanosome and human TFIIH. While the ring-shaped core domain was surprisingly congruent between the two structures, trypanosome TFIIH lacked the knob-like CAK moiety and exhibited extra densities on either side of the ring, presumably due to the specific subunits