Promoter orientation and sequence determine which DNA strand is transcribed by RNA polymerase
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Reference literature and encyclopedia entries establish that promoter sequences and their orientation determine transcription initiation sites and direct RNA polymerase to transcribe specific DNA strands.
Promoter escape is the last stage of transcription initiation when RNA polymerase, having initiated de novo phosphodiester bond synthesis, must begin to relinquish its hold on promoter DNA and advance to downstream regions (DSRs) of the template. In vitro, this process is marked by the release of high levels of abortive transcripts at most promoters, reflecting the high instability of initial transcribing complexes (ITCs) and indicative of the existence of barriers to the escape process. The high abortive initiation level is the result of the existence of unproductive ITCs that carry out repeated initiation and abortive release without escaping the promoter. The formation of unproductive ITCs is a widespread phenomenon, but it occurs to different extent on different promoters. Quantitative analysis of promoter mutations suggests that the extent and pattern of abortive initiation and promoter escape is determined by the sequence of promoter elements, both in the promoter recognition region (PRR) and the initial transcribed sequence (ITS). A general correlation has been found that the stronger the promoter DNA-polymerase interaction, the poorer the ability of RNA polymerase to escape the promoter. In gene regulation, promoter escape can be the rate-limiting step for transcription initiation. An increasing number of regulatory proteins are known to exert their control at this step. Examples are discussed with an emphasis on the diverse mechanisms involved. At the molecular level, the X-ray crystal structures of RNA polymerase and its various transcription complexes provide the framework for understanding the functional data on abortive initiation and promoter escape. Based on structural and biochemical evidence, a mechanism for abortive initiation and promoter escape is described.
Abstract Guanine-rich (G-rich) homopurine–homopyrimidine nucleotide sequences can block transcription with an efficiency that depends upon their orientation, composition and length, as well as the presence of negative supercoiling or breaks in the non-template DNA strand. We report that a G-rich sequence in the non-template strand reduces the yield of T7 RNA polymerase transcription by more than an order of magnitude when positioned close (9 bp) to the promoter, in comparison to that for a distal (∼250 bp) location of the same sequence. This transcription blockage is much less pronounced for a C-rich sequence, and is not significant for an A-rich sequence. Remarkably, the blockage is not pronounced if transcription is performed in the presence of RNase H, which specifically digests the RNA strands within RNA–DNA hybrids. The blockage also becomes less pronounced upon reduced RNA polymerase concentration. Based upon these observations and those from control experiments, we conclude that the blockage is primarily due to the formation of stable RNA–DNA hybrids (R-loops), which inhibit successive rounds of transcription. Our results could be relevant to transcription dynamics in vivo (e.g. transcription ‘bursting’) and may also have practical implications for the design of expression vectors.
Abstract Promoter escape involves breaking of the favourable contacts between RNA polymerase (RNAP) and the promoter to allow transition to an elongation complex. The sequence of DNA template that is transcribed during promoter escape (ITS; Initially Transcribed Sequence) can affect promoter escape by mechanisms that are not yet fully understood. We employed a highly parallel strategy utilizing Next Generation Sequencing (NGS) to collect data on escape properties of thousands of ITS variants. We show that ITS controls promoter escape through a combination of position-dependent effects (most prominently, sequence-directed RNAP pausing), and position-independent effects derived from sequence encoded physical properties of the template (for example, RNA/DNA duplex stability). ITS often functions as an independent unit affecting escape in the same manner regardless of the promoter from which transcription initiates. However, in some cases, a strong dependence of ITS effects on promoter context was observed suggesting that promoters may have ‘allosteric’ abilities to modulate ITS effects. Large effects of ITS on promoter output and the observed interplay between promoter sequence and ITS effects suggests that the definition of bacterial promoter should include ITS sequence.
We report the identification of DNA sequences that determine the activity of the Streptomyces galP1 promoter and a new form of RNA polymerase holoenzyme that recognizes these sequences in vitro. Base substitutions were introduced throughout the galP1 promoter region, and bases at positions -34, -36, and -11 with respect to the transcription start site were shown to be required for promoter function. These bases correspond in their positions to regions known to be important for RNA polymerase binding in several classes of eubacterial promoters, but the sequences themselves are not similar to those previously described. The -35 region of the galP1 promoter consists of six G residues, and base changes in this G hexamer had a dramatic effect on promoter activity. By using galP1-containing DNA template, a new RNA polymerase activity was purified from Streptomyces. Holoenzyme reconstitution experiments identified a new sigma factor that directs galP1 transcription in vitro. DNase I protection experiments identified a binding site for this new holoenzyme immediately upstream of the galP1 transcription start site.
Recognition of promoters in bacterial RNA polymerases (RNAPs) is controlled by sigma subunits. The key sequence motif recognized by the sigma, the −10 promoter element, is located in the non-template strand of the double-stranded DNA molecule ~10 nucleotides upstream of the transcription start site. Here, we explain the mechanism by which the phage AR9 non-virion RNAP (nvRNAP), a bacterial RNAP homolog, recognizes the −10 element of its deoxyuridine-containing promoter in the template strand. The AR9 sigma-like subunit, the nvRNAP enzyme core, and the template strand together form two nucleotide base-accepting pockets whose shapes dictate the requirement for the conserved deoxyuridines. A single amino acid substitution in the AR9 sigma-like subunit allows one of these pockets to accept a thymine thus expanding the promoter consensus. Our work demonstrates the extent to which viruses can evolve host-derived multisubunit enzymes to make transcription of their own genes independent of the host. Promoter recognition is a critical step in the initiation of transcription of DNA to RNA. Here, the authors describe a novel mechanism by which a phage-encoded RNA polymerase recognizes viral promoters containing deoxyuridines instead of thymidines.
How DNA sequence affects the dynamics and position of RNA Polymerase II (Pol II) during transcription remains poorly understood. Here, we used naturally occurring genetic variation in F1 hybrid mice to explore how DNA sequence differences affect the genome-wide distribution of Pol II. We measured the position and orientation of Pol II in eight organs collected from heterozygous F1 hybrid mice using ChRO-seq. Our data revealed a strong genetic basis for the precise coordinates of transcription initiation and promoter proximal pause, allowing us to redefine molecular models of core transcriptional processes. Our results implicate DNA sequence, including both known and novel DNA sequence motifs, as key determinants of the position of Pol II initiation and pause. We report evidence that initiation site selection follows a stochastic process similar to Brownian motion along the DNA template. We found widespread differences in the position of transcription termination, which impact the primary structure and stability of mature mRNA. Finally, we report evidence that allelic changes in transcription often affect mRNA and ncRNA expression across broad genomic domains. Collectively, we reveal how DNA sequences shape core transcriptional processes at single nucleotide resolution in mammals.
A small non-histone protein of budding yeast, Nhp6 has been reported to specifically influence the transcription of a yeast gene, SNR6. The gene is essential, transcribed by the enzyme RNA polymerase III, and codes for the U6snRNA required for mRNA splicing. A translationally positioned nucleosome on the gene body enables the assembly factor TFIIIC binding by juxtaposing its otherwise widely separated binding sites, boxes A and B. We found histone depletion results in the loss of U6 snRNA production. Changing the rotational phase of the boxes and the linear distance between them with deletions in 5 bp steps displayed a helical periodicity in transcription, which gradually reduced with incremental deletions up to 40 bp but increased on further deletions enclosing the pseudoA boxes. Nhp6 influences the transcription in a dose-dependent manner, which is modulated by its previously reported co-operator, an upstream stretch of seven T residues centered between the TATA box and transcription start site. Nhp6 occupancy on the gene in vivo goes up at least 2-fold under the repression conditions. Nhp6 absence, T7 disruption, or shorter A–B box distance all cause the downstream initiation of transcription. The right +1 site is selected with the correct placement of TFIIIC before the transcription initiation factor TFIIIB. Thus, the T7 sequence and Nhp6 help the assembly and placement of the transcription complex at the right position. Apart from the chromatin remodelers, the relative r
a promoter is a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter. The
In genetics, a promoter is a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter. The RNA transcript may encode a protein (mRNA), or can have a function in and of itself, such as tRNA or rRNA. Promoters are located near the transcription start sites of genes, upstream on the DNA (towards the 5' region of the sense stra
In genetics, a promoter is a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter. The RNA transcript may encode a protein (mRNA), or can have a function in and of itself, such as tRNA or rRNA. Promoters are located near the transcription start sites of genes,…
The primary regulators of metazoan gene expression are enhancers, originally functionally defined as DNA sequences that can activate transcription at promoters in an orientation-independent and distance-independent manner. Despite being crucial for gene regulation in animals, what mechanisms underlie enhancer selectivity for promoters, and more fundamentally, how enhancers interact with promoters and activate transcription, remain poorly understood. In this Review, we first discuss current models of enhancer-promoter interactions in space and time and how enhancers affect transcription activation. Next, we discuss different mechanisms that mediate enhancer selectivity, including repression, biochemical compatibility and regulation of 3D genome structure. Through 3D polymer simulations, we illustrate how the ability of 3D genome folding mechanisms to mediate enhancer selectivity strongly varies for different enhancer-promoter interaction mechanisms. Finally, we discuss how recent technical advances may provide new insights into mechanisms of enhancer-promoter interactions and how technical biases in methods such as Hi-C and Micro-C and imaging techniques may affect their interpretation.
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