Faithful Transcription Research Articles

A critical response to Halley's (2022) ‘Audubon’s diary transcripts were doctored to support his false claim of personally discovering Lincoln’s Sparrow Melospiza lincolnii (Audubon, 1834)’

In a recent article (Bull. Brit. Orn. Cl. 142: 329–342), Matthew Halley contended that John James Audubon (1785–1851) lied about his discovery of Lincoln's Sparrow Melospiza lincolnii (Audubon, 1834) during his 1833 Labrador expedition. Extracts from the naturalist's journal, published after his death in a biography prepared by his widow, Lucy (1787–1874), states that he was aboard ship ‘Drawing all day’ when the specimen was collected by one of his assistants. Consequently, Halley submitted that Audubon’s claim in the Ornithological biography to having first sighted the bird was fabricated and that his granddaughter Maria R. Audubon (1843–1925) doctored her alternate version of the journal to be consistent before she destroyed the original. However, Halley overlooked critical facts, including evidence that Lucy’s manuscript was compiled and edited by others; the published work contained numerous errors; and the journal entries for the previous two weeks were misdated and sometimes conjoined from multiple days, proving that her journal was not a faithful transcription of the original.

Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs.

To synthesize a chirally inverted ribosome with the goal of building mirror-image biology systems requires the preparation of kilobase-long mirror-image ribosomal RNAs that make up the structural and catalytic core and about two-thirds of the molecular mass of the mirror-image ribosome. Here, we chemically synthesized a 100-kilodalton mirror-image T7 RNA polymerase, which enabled efficient and faithful transcription of the full-length mirror-image 5S, 16S, and 23S ribosomal RNAs from enzymatically assembled long mirror-image genes. We further exploited the versatile mirror-image T7 transcription system for practical applications such as biostable mirror-image riboswitch sensor, long-term storage of unprotected kilobase-long l-RNA in water, and l-ribozyme-catalyzed l-RNA polymerization to serve as a model system for basic RNA research.

NF-Y controls fidelity of transcription initiation at gene promoters through maintenance of the nucleosome-depleted region

Faithful transcription initiation is critical for accurate gene expression, yet the mechanisms underlying specific transcription start site (TSS) selection in mammals remain unclear. Here, we show that the histone-fold domain protein NF-Y, a ubiquitously expressed transcription factor, controls the fidelity of transcription initiation at gene promoters in mouse embryonic stem cells. We report that NF-Y maintains the region upstream of TSSs in a nucleosome-depleted state while simultaneously protecting this accessible region against aberrant and/or ectopic transcription initiation. We find that loss of NF-Y binding in mammalian cells disrupts the promoter chromatin landscape, leading to nucleosomal encroachment over the canonical TSS. Importantly, this chromatin rearrangement is accompanied by upstream relocation of the transcription pre-initiation complex and ectopic transcription initiation. Further, this phenomenon generates aberrant extended transcripts that undergo translation, disrupting gene expression profiles. These results suggest NF-Y is a central player in TSS selection in metazoans and highlight the deleterious consequences of inaccurate transcription initiation.

UVB induces a genome-wide acting negative regulatory mechanism that operates at the level of transcription initiation in human cells.

Faithful transcription of DNA is constantly threatened by different endogenous and environmental genotoxic effects. Transcription coupled repair (TCR) has been described to stop transcription and quickly remove DNA lesions from the transcribed strand of active genes, permitting rapid resumption of blocked transcription. This repair mechanism has been well characterized in the past using individual target genes. Moreover, numerous efforts investigated the fate of blocked RNA polymerase II (Pol II) during DNA repair mechanisms and suggested that stopped Pol II complexes can either backtrack, be removed and degraded or bypass the lesions to allow TCR. We investigated the effect of a non-lethal dose of UVB on global DNA-bound Pol II distribution in human cells. We found that the used UVB dose did not induce Pol II degradation however surprisingly at about 93% of the promoters of all expressed genes Pol II occupancy was seriously reduced 2–4 hours following UVB irradiation. The presence of Pol II at these cleared promoters was restored 5–6 hours after irradiation, indicating that the negative regulation is very dynamic. We also identified a small set of genes (including several p53 regulated genes), where the UVB-induced Pol II clearing did not operate. Interestingly, at promoters, where Pol II promoter clearance occurs, TFIIH, but not TBP, follows the behavior of Pol II, suggesting that at these genes upon UVB treatment TFIIH is sequestered for DNA repair by the TCR machinery. In agreement, in cells where the TCR factor, the Cockayne Syndrome B protein, was depleted UVB did not induce Pol II and TFIIH clearance at promoters. Thus, our study reveals a UVB induced negative regulatory mechanism that targets Pol II transcription initiation on the large majority of transcribed gene promoters, and a small subset of genes, where Pol II escapes this negative regulation.

Cancionero: Manuscrito 1250 de la Biblioteca del Palacio Real by Gómez Manrique (review)

Reviewed by: Cancionero: Manuscrito 1250 de la Biblioteca del Palacio Real by Gómez Manrique Connie L. Scarborough Gómez Manrique. Cancionero: Manuscrito 1250 de la Biblioteca del Palacio Real. Ed. José I. Suárez. New York: National Hispanic Foundation for the Humanities, 2013. 596p. José Suárez has provided scholars of fifteenth-century Spanish letters with a new and very faithful transcription of the cancionero of one of the most important men “of arms and letters” of the period—Gómez Manrique. Manuscript 1250, [End Page 171] housed at the Royal Palace in Madrid, is considered the most complete collection of Gómez Manrique’s poetry, but, surprisingly, it has never heretofore been transcribed in its entirety. Beyond giving us the complete transcription of the manuscript, Suárez meticulously documents instances where versions of poems included in ms. 1250 differ from those found in other fragmentary collections of Manrique’s poetry. This is not a critical edition per se, but a transcription that maintains the manuscript’s original orthography and its present pagination. Manuscript 1250 was prepared for, and dedicated to, don Rodrigo Pimentel, Conde de Benavente, a relative of the poet’s mother, and its date of composition is sometime after 1476. As such it does not include all of the known poems authored by Manrique since some only appear in other manuscripts or were authored after the production of ms. 1250 and, obviously, before Manrique’s death in ca 1491. To supplement the materials contained in ms. 1250, Suárez provides a useful appendix of all the poet’s known compositions that do not appear in it. Suárez gives variants for ms. 1250 by comparing its compositions with nine other manuscripts containing Manrique’s poetry as well as versions published in 1928 in the “Nueva Biblioteca de Autores Españoles” (NBAE) series, vol. 4. The inclusion of variants is particularly useful for researchers who rarely have the opportunity to consult all the manuscripts consulted by Suárez. Also helpful are appendices that classify the poems by subject matter: love, politics/morals, events, satire, religion, and drama as well as an appendix of first lines. Since Manrique’s work is extant in various manuscripts scattered throughout collections in Spain, including the Biblioteca Nacional (Madrid), Palacio Real (Madrid), and the Biblioteca de Menéndez Pelayo (Santander), it is especially helpful to be able to consult variants in one convenient publication. This is the most comprehensive edition of Gómez Manrique’s Cancionero since Antonio Paz y Mélia’s 1885 compilation. Obviously, the nineteenth-century edition prepared by Paz y Mélia does not adhere to the current, rigorous standards for manuscript transcription employed by Suárez. Also, maintaining the pagination of the manuscript for this transcription greatly facilitates locating passages when scholars need to consult the original. The book itself is attractively printed with a very clear typeface and accessibly priced. [End Page 172] Connie L. Scarborough Texas Tech University Copyright © 2013 Rocky Mountain Modern Language Association

Césaire and the Challenge of Translation: The Example of “Strong Men” by Sterling Brown

Césaire and the Challenge of Translation: The Example of “Strong Men” by Sterling Brown

Importance of Steric Effects on the Efficiency and Fidelity of Transcription by T7 RNA Polymerase

DNA-dependent RNA polymerases such as T7 RNA polymerase (T7 RNAP) perform the transcription of DNA into mRNA with high efficiency and high fidelity. Although structural studies have provided a detailed account of the molecular basis of transcription, the relative importance of factors like hydrogen bonds and steric effects remains poorly understood. We report herein the first study aimed at systematically probing the importance of steric and electrostatic effects on the efficiency and fidelity of DNA transcription by T7 RNAP. We used synthetic nonpolar analogues of thymine with sizes varying in subangstrom increments to probe the steric requirements of T7 RNAP during the elongation mode of transcription. Enzymatic assays with internal radiolabeling were performed to compare the efficiency of transcription of modified DNA templates with a natural template containing thymine as a reference. Furthermore, we analyzed effects on the fidelity by measuring the composition of RNA transcripts by enzymatic digestion followed by two-dimensional thin layer chromatography separation. Our results demonstrate that hydrogen bonds play an important role in the efficiency of transcription but, interestingly, do not appear to be required for faithful transcription. Steric effects (size and shape variations) are found to be significant both in insertion of a new RNA base and in extension beyond it.

Chromatin remodelling: why, when & how?

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The Glorified Woman: Abstraction and Domination in Le Livre du voir dit

Pour que je puisse dire: cette femme, il faut que d'une maniere ou d'une autre je lui retire sa realite d'os et de chair, la rende absente et l'aneantisse. Le mot me donne l'etre, mais il me donne prive d'etre. --Maurice Blanchot, La Part du feu On the surface, Guillaume de Machaut's Le Livre du voir dit (1362-65) is the transcription and description of an actual love affair supposedly occurring between Machaut anda beautiful young noblewoman whom he calls Toute Belle. Machaut's self-stated goal as author is to be as inclusive as possible in order to render a truthful account of what happened between the lovers, providing the reader with, as Jacqueline Cerquiglini-Toulet puts it: tout: le noir et blanc, l'amour et desamour (Introduction 21). To this end, Le Livre du voir dit is a sprawling, polyphonic collection of reproduced letters and verse supposedly written between Machaut and the noblewoman in real life and then transcribed in the Voir dit, where a narrative supplied by Machaut accompanies the transcriptions. The reader follows along as the purely epistolary relationship becomes a physical one. After some tensions caused by the rarity of actual visits and by rumors of infidelity, the lovers are ultimately portrayed as having achieved an ideal courtly union. Ostensibly, then, the Voir dit (True Poem) is merely the faithful transcription of what happened between two real-life lovers. However, as Blanchot reminds us, reality cannot be transcribed into language without violence. In the case of the Voir dit, this violence is exacerbated (and highlighted) because the discourse of the text--that of medieval courtly verse--is innately infused with ah ideology (that of fin'amors) according to which Guillaume must be cast as the valiant, unwavering lover and Toute Belle--as indicated by the name--must incarnate pure beauty (along with other traits of purity). Numerous studies have demonstrated to what extent the medieval cleric, as writer, could betray a desire to create for himself, among his secular peers, an impressive persona through literature, this has been shown to be the case for Richard de Fournival, (1) Abelard, (2) and, as Kevin Brownlee and Deborah McGrady suggest, for Guillaume de Machaut himself. (3) So we should not be surprised if Guillaume's (4) lofty verse and exahed descriptions of the love affair tend to paint a rather glorious and even allegorical portrait of Machaut in the guise of giving a truthful account. However, when we consider the consequences of this glorification for the called Toute Belle, who must also have an allegorical other imposed upon her, we start to glimpse an economy of domination that underlies Guillaume's retelling of the love affair. Tapping into that economy and describing it will allow us to move beyond limitations of previous scholarship concerning the Voir dit, limitations that--while discerning ulterior motives in Machaut's depiction of the love affair (i.e., building his own image)--have ignored the violence done to the the text must sacrifice to realize those motives. In Controlling Readers, McGrady has pointed to the largely ignored role (46) of Guillaume's inscribed audience in the Voir dit--invasive in terms of its capacity to disrupt and alter the course of Guillaume's literary project. She includes Toute Belle in this audience. However, such an invasive presence is more accurately described as coming from the marginal voice of the behind the construct of Toute Belle; we must distinguish between the two, because Toute Belle is perfectly congruent and necessary to Guillaume's literary project and therefore incapable of being invasive. Guillaume would write the perfect at the expense of the authentic, imperfect we glimpse in the margins of the Voir dit. It should be emphasized here that by authentic woman I do not refer to someone having existed outside of the text (this paper does not participate in that debate). …

A social orthography of identity: the N'ko literacy movement in West Africa

This article explores the development and spread of the N'ko script among Mande communities in West Africa. N'ko presents a rare example of an indigenous script that has successfully competed against other writing systems that are older, better financed, and propagated in religious and formal education. N'ko script is studied in relation to its role as one of the most popular and widespread indigenous scripts in contemporary West Africa. The social relevance of N'ko literacy is contrasted with colonial and national literacy education programs. N'ko's popularity is shown to result from the script's strong linguistic and cultural relevance to Mande communities through its faithful transcription of local languages and its corpus of publications on indigenous and foreign knowledge. The introduction of formal schooling in N'ko is analyzed as a significant recent shift in the literacy movement that presents new opportunities and challenges. The internationalization of the movement is shown to have strengthened support for N'ko literacy, with N'ko serving as an important contemporary symbol of Mande social identity, which the author terms its “social orthography.” However, N'ko's strong association with Mande identity also threatens to limit the literacy movement's future development.

Faithful transcription initiation from a mitochondrial promoter in transgenic plastids

The transcriptional machineries of plastids and mitochondria in higher plants exhibit striking similarities. All mitochondrial genes and part of the plastid genes are transcribed by related phage-type RNA polymerases. Furthermore, the majority of mitochondrial promoters and a subset of plastid promoters show a similar structural organization. We show here that the plant mitochondrial atpA promoter is recognized by plastid RNA polymerases in vitro and in vivo. The Arabidopsis phage-type RNA polymerase RpoTp, an enzyme localized exclusively to plastids, was found to recognize the mitochondrial atpA promoter in in vitro assays suggesting the possibility that mitochondrial promoters might function as well in plastids. We have, therefore, generated transplastomic tobacco plants harboring in their chloroplast genome the atpA promoter fused to the coding region of the bacterial nptII gene. The chimeric nptII gene was found to be efficiently transcribed in chloroplasts. Mapping of the 5′ ends of the nptII transcripts revealed accurate recognition of the atpA promoter by the chloroplast transcription machinery. We show further that the 5′ untranslated region (UTR) of the mitochondrial atpA transcript is capable of mediating translation in chloroplasts. The functional and evolutionary implications of these findings as well as possible applications in chloroplast genome engineering are discussed.

Carlos Poveda's Menu

Carlos Poveda's Domestic Landscapes are linked to a history of food and art that reaches back to Greco-Roman antiquity and becomes empowered with contemporary artists who sculpt or paint their works in edible materials to be devoured by spectators. Poveda's Landscapes, however, offer food that is symbolic——inedible. He reinvents the organic by using industrial refuse that he converts, colors, and models in a cauldron in a process as akin to alchemy as to cooking. His is not a faithful transcription of meals in the style of classical still lifes, but rather an artistic overlapping of emotions, that surround the idea of the edible. Looking at his sculptures we may feel revulsion, but what sickens us is not so much his creation as the awareness it brings of our intrinsically predatory nature. He gives us an art form that not only fails to provoke appetite but also touches our deepest culinary memories and leads us back to a primal past by asserting the significance of food in our collective memory. Ultimately, our strongest reaction to his work may be the fear that we won't be able to digest the absurdity of our daily life.

Self-Correction Ensures Faithful Transcription

Self-Correction Ensures Faithful Transcription

The mitochondrion as a primary site of action of steroid and thyroid hormones: Presence and action of steroid and thyroid hormone receptors in mitochondria of animal cells

Mitochondria are key cellular organelles that regulate events related to energy production and apoptosis. These processes are modulated, in turn, by steroid and thyroid hormones in the course of their actions on metabolism, growth and development. In this context, a direct effect of these hormones on the mitochondrial-linked processes, possibly by way of cognate mitochondrial receptors, has been proposed. In this paper we review data from the literature and present new findings supporting this concept. Receptors for steroid hormones, glucocorticoids and estrogens, and for T 3, have been detected in mitochondria by immunofluorescence labeling and confocal laser microscopy, by Western blotting of mitochondrial proteins and by immunogold electron microscopy. Furthermore, the mitochondrial genome contains nucleotide sequences with high similarity to known hormone-responsive elements, which interact with the appropriate receptors to confer hormone-dependent activation of reporter genes in transfection experiments. Thus, thyroid hormone stimulates mitochondrial transcription mediated by the cognate receptor when added to an in organello mitochondrial system, capable of faithful transcription.

The role of simian virus 5 V protein on viral RNA synthesis

The paramyxovirus simian virus 5 (SV5) has seven genes but encodes eight known viral proteins. The V/P gene is transcribed into two mRNA species: V mRNA from a faithful transcription of the gene and P mRNA from transcription with addition of two G residues at a specific site of the gene. V, a 222-amino acid (AA) residue protein, and P, a 392 AA residue protein, share an identical N-terminus domain of 164 amino acid residues. P is essential for SV5 RNA replication and transcription. Whereas it is known that V plays important roles in virus pathogenesis, the role of V in SV5 replication and transcription is not clear. A mini-genome system, free of vaccinia virus gene expression system, consisting of plasmids expressing NP, P, and L, as well as a plasmid encoding a reporter gene, chloramphenicol acetyltransferase (CAT) flanked by SV5 trailer and leader sequences under control of a bacteriophage T7 RNA polymerase promoter, has been established to examine the role of V in SV5 RNA transcription and replication. Addition of V-expressing plasmid in the mini-genome system caused inhibition of the reporter gene expression, suggesting that V plays a role in regulating SV5 gene expression. By examining the amount of encapsidated viral RNA genome using reverse transcription with primer annealing to viral anti-genome RNA and PCR, it was found that expression of V reduced the amount of viral RNA genome in the mini-genome system, suggesting that V inhibits viral RNA replication. To examine whether the V protein inhibits viral RNA transcription as well, a mini-genome system with a defective anti-genome promoter (AGP) such that a reporter gene (luciferase, Luc) expression is only derived from transcription of newly produced mini-genome and not from de novo replicated viral genome due to the defect in replication element has been utilized. The V protein inhibited luciferase expression from the mini-genome with a defective AGP, suggesting V inhibits SV5 transcription. Thus, SV5 V inhibits both SV5 RNA replication and transcription.

TFIIIC-independent in vitro transcription of yeast tRNA genes

The most peculiar transcriptional property of eukaryotic tRNA genes, as well as of other genes served by RNA polymerase III, is their complete dependence on the intragenic interaction platform provided by transcription factor IIIC (TFIIIC) for the productive assembly of the TBP-containing initiation factor TFIIIB. The sole exception, in yeast, is the U6 RNA gene, which is able to exploit a TATAAATA element, 30 bp upstream of the transcription start site, for the TFIIIC-independent assembly of TFIIIB. To find out whether this extragenic core promoter organization and autonomous TFIIIB assembly capacity are unique features of the U6 gene or also apply to other genes transcribed by RNA polymerase III, we scanned the 5′-flanking regions (up to position −100) of the entire tRNA gene set of Saccharomyces cerevisiae searching for U6-like TATA motifs. Four tRNA genes harboring such a sequence motif around position −30 were identified and found to be transcribed in vitro by a minimal system only composed of TFIIIB and RNA polymerase III. In this system, start site selection is not at all affected by the absence of TFIIIC, which, when added, significantly stimulates transcription by determining an increase in the number, rather than in the efficiency of utilization, of productive initiation complexes. A specific TBP-TATA element interaction is absolutely required for TFIIIC-independent transcription, but the nearby sequence context also contributes to the efficiency of autonomous TFIIIB assembly. The existence of a TFIIIB assembly pathway leading to the faithful transcription of natural eukaryotic tRNA genes in the absence of TFIIIC provides novel insights into the functional flexibility of the eukaryotic tRNA gene transcription machinery and on its evolution from an ancestral RNA polymerase III system relying on upstream, TATA-centered control elements.

Functional Characterization of ABC10α, an Essential Polypeptide Shared by All Three Forms of Eukaryotic DNA-dependent RNA Polymerases

ABC10alpha is a small polypeptide shared by the three yeast RNA polymerases. Homologous polypeptides in higher eukaryotes have a zinc-binding CX(2)C. CX(2)C motif and a conserved basic C-terminal end. These features are also found in archaeal gene products that may encode an RNA polymerase subunit. The CX(2)C. CX(2)C motif is partly dispensable, since only its first cysteine is essential for growth, whereas the basic C-terminal end is critical in vivo. A mutant in the latter domain has an RNA polymerase III-specific defect and, in vitro, impairs RNA polymerase III assembly. Polymerase activity was, however, not affected in various faithful transcription assays. The mutant is suppressed by a high gene dosage of the second largest subunit of RNA polymerase III, whereas the homologous subunits of RNA polymerase I and II have aggravating effects. In a two-hybrid assay, ABC10alpha binds to the C-terminal half of the second largest subunit of RNA polymerase I, in a way that requires the integrity of the CX(2)C. CX(2)C motif. Thus, ABC10alpha appears to interact directly with the second largest subunit during polymerase assembly. This interaction is presumably a major rate-limiting step in assembly, since diploid cells containing only one functional gene copy for ABC10alpha have a partial growth defect.

A spider tRNA(Ala) requires a far upstream sequence element for expression.

Within the series of timed differential accumulations of small RNAs we have shown to prelude the synthesis of fibroin in the large ampullate glands of Nephila clavipes (Nc), we are currently directing our attention to the alanine tRNAs. This work reports the subcloning of the members of a tRNAAla gene cluster and the optimization of their transcription in a heterologous cell-free system derived from Bombyx mori (Bm) silkglands. Our data show that the heterologous cell-free system supports the faithful and differential transcription of the individual spider alanine tRNA genes. We are thus making use of the extract to characterize the individual genes with respect to flank-contained regulatory elements through cell-free transcription of gene derivatives. The work has been initiated with pNTA3 because of its high transcriptional activity. Interestingly, the transcription of this gene requires a far upstream sequence, an uncommon modality in tRNA genes.

PTK, the chloroplast RNA polymerase-associated protein kinase from mustard (Sinapis alba), mediates redox control of plastid in vitro transcription.

The major RNA polymerase from mustard chloroplasts is a multi-subunit enzyme consisting of core components and associated factors. Among the latter is a heterotrimeric factor named PTK (plastid transcription kinase) because of its serine/threonine-type protein kinase activity. PTK activity itself depends on its phosphorylation state. In addition, we show that it responds to glutathione but not to other redox-reactive reagents that were tested, and both glutathione and phosphorylation act antagonistically. Using a homologous in vitro system, we find that PTK selectively phosphorylates subunit(s) of plastid RNA polymerase and is involved in determining the level of faithful transcription from the chloroplast psbA promoter. Together, these results establish a role for phosphorylation and redox state in the regulation of plastid gene expression.

Transcription of the juvenile hormone esterase gene under the control of both an initiator and AT-rich motif.

The binding of transcription factors to the core promoter of the juvenile hormone esterase gene was functionally characterized using both a cell-free in vitro transcription functional assay and a cell transfection assay. A core JHE promoter (-61 to +28 bp relative to transcription start site) supported faithful transcription from the in vivo transcription start site. The nuclear extracts from the Sf9 insect cell line that provided transcription from that template also bound to that template as a probe in gel-mobility shift assays. Deletion or transversion of the initiator-binding motif (-1 to +4 bp) abolished detectable transcription either in vitro or in transfected cells. An AT-rich motif (ATATAT; -28 to -23 bp) serves another transcription factor-binding site. Mutation of the AT-rich motif to a canonical TATA-box preserved transcription, while either its deletion or complete transversion abolished or significantly reduced detectable transcriptional activity. These results indicate that, under these conditions, the functional operation of this core promoter approaches that of a composite promoter in which both the TATA- and initiator-binding protein complexes are necessary, even for basal transcription. On the other hand, these debilitating mutations to either the TATA box or initiator motif did not prevent the ability of the corresponding gel-shift competitive probes to compete with the wild-type promoter for binding by the transcription factors. Even a double transversion of both the AT-rich motif and the initiator-binding motif was able to competitively displace the protein complex that bound to the labelled wild-type probe. These data strongly indicate the presence of (an) additional core-promoter-associated transcription factor(s) (that is not the 'downstream element') that contact(s) the AT-binding complex and/or initiator-binding factor with sufficient avidity to remove them from binding to the competing wild-type promoter sequence.