SummaryThe RNA polymerase II core promoter is generally defined to be the sequence that directs the initiation of transcription. This simple definition belies a diverse and complex transcriptional module. There are two major types of core promoters -focused and dispersed. Focused promoters contain either a single transcription start site or a distinct cluster of start sites over several nucleotides, whereas dispersed promoters contain several start sites over 50 to 100 nucleotides and are typically found in CpG islands in vertebrates. Focused promoters are more ancient and widespread throughout nature than dispersed promoters; however, in vertebrates, dispersed promoters are more common than focused promoters. In addition, core promoters may contain many different sequence motifs, such as the TATA box, BRE, Inr, MTE, DPE, DCE, and XCPE1, that specify different mechanisms of transcription and responses to enhancers. Thus, the core promoter is a sophisticated gateway to transcription that determines which signals will lead to transcription initiation.

The TCT motif (polypyrimidine initiator) encompasses the transcription start site of nearly all ribosomal protein genes in Drosophila and mammals. The TCT motif is required for transcription of ribosomal protein gene promoters. The TCT element resembles the Inr (initiator), but is not recognized by TFIID and cannot function in lieu of an Inr. However, a single T-to-A substitution converts the TCT element into a functionally active Inr. Thus, the TCT motif is a novel transcriptional element that is distinct from the Inr. These findings reveal a specialized TCT-based transcription system that is directed toward the synthesis of ribosomal proteins.Supplemental material is available at http://www.genesdev.org.

The RNA polymerase II core promoter is a diverse and complex regulatory element. To gain a better understanding of the core promoter, we examined the motif 10 element (MTE), which is located downstream of the transcription start site and acts in conjunction with the initiator (Inr). We found that the MTE promotes the binding of purified TFIID to the core promoter and that the TAF6 and TAF9 subunits of TFIID appear to be in close proximity to the MTE. To identify the specific nucleotides that contribute to MTE activity, we performed a detailed mutational analysis and determined a functional MTE consensus sequence. These studies identified favored as well as disfavored nucleotides and demonstrated the previously unrecognized importance of nucleotides in the subregion of nucleotides 27 to 29 (؉27 to ؉ 29 relative to A ؉1 in the Inr consensus) for MTE function. Further analysis led to the identification of three downstream subregions (nucleotides 18 to 22, 27 to 29, and 30 to 33) that contribute to core promoter activity. The three binary combinations of these subregions lead to the MTE (nucleotides 18 to 22 and 27 to 29), a downstream core promoter element (nucleotides 27 to 29 and 30 to 33), and a novel "bridge" core promoter motif (nucleotides 18 to 22 and 30 to 33). These studies have thus revealed a tripartite organization of key subregions in the downstream core promoter.The expression of the tens of thousands of genes within a cell is regulated during growth, development, and response to environmental stimuli. In eukaryotes, transcription of proteincoding genes is mediated by RNA polymerase II. Transcription by RNA polymerase II is regulated by a wide variety of factors that include the basal transcription factors, sequence-specific enhancer-and promoter-binding proteins, coregulatory factors, and other chromatin remodeling and modifying factors. The signals from these factors ultimately converge at the core promoter during the process of transcription initiation (for reviews, see references 7, 12, 21, and 23).The RNA polymerase II core promoter is the region of DNA that directs the initiation of transcription and generally spans from about nucleotide Ϫ40 to ϩ40 relative to the transcription start site. The core promoter is diverse in terms of its structure and function as there are different mechanisms by which RNA polymerase II can be recruited to the promoter. For transcription that is directed by the TFIID transcription factor, there are several known sequences, termed core promoter elements, that mediate the recruitment of TFIID as well as other basal transcription factors to the DNA template. These core promoter elements include the TATA box, the initiator (Inr), the motif 10 element (MTE), the downstream core promoter element (DPE), the TFIIB recognition elements (BRE u and BRE d ), the downstream core element (DCE), and the X core promoter element 1 (XCPE1) (for a recent review, see reference 12). In this work, we focus on the analysis of the MTE.The study of the MTE began with the identification of m...

Early detection of infection with SARS-CoV-2 is key to managing the current global pandemic, as evidence shows the virus is most contagious on or before symptom onset 1,2 . Here, we introduce a low-cost, high-throughput method for diagnosis of SARS-CoV-2 infection, dubbed Pathogen-Oriented Low-Cost Assembly & Re-Sequencing (POLAR), that enhances sensitivity by aiming to amplify the entire SARS-CoV-2 genome rather than targeting particular viral loci, as in typical RT-PCR assays. To achieve this goal, we combine a SARS-CoV-2 enrichment method developed by the ARTIC Network (https://artic.network/) with short-read DNA sequencing and de novo genome assembly. We are able to reliably (>95% accuracy) detect SARS-CoV-2 at concentrations of 84 genome equivalents per milliliter, better than the reported limits of detection of almost all diagnostic methods currently approved by the US Food and Drug Administration. At higher concentrations, we are able to reliably assemble the SARS-CoV-2 genome in the sample, often with no gaps and perfect accuracy. Such genome assemblies enable the spread of the disease to be analyzed much more effectively than would be possible with an ordinary yes/no diagnostic, and can help identify vaccine and drug targets. Using POLAR, a single person can process 192 samples over the course of an 8-hour experiment, at a cost of ~$30/patient, enabling a 24-hour turnaround with sequencing and data analysis time included. Further testing and refinement will likely enable greater enhancements in the sensitivity of the above approach.