Engineering efficient termination of bacteriophage T7 RNA polymerase transcription

The bacteriophage T7 expression system is one of the most prominent transcription systems used in biotechnology and molecular-level research. However, T7 RNA polymerase is prone to read-through transcription due to its high processivity. As a consequence, enforcing efficient transcriptional termination is difficult. The termination hairpin found natively in the T7 genome is adapted to be inefficient, exhibiting 62% termination efficiency in vivo and even lower efficiency in vitro. In this study, we engineered a series of sequences that outperform the efficiency of the native terminator hairpin. By embedding a previously discovered 8-nucleotide T7 polymerase pause sequence within a synthetic hairpin sequence, we observed in vivo termination efficiency of 91%; by joining two short sequences into a tandem 2-hairpin structure, termination efficiency was increased to 98% in vivo and 91% in vitro. This study also tests the ability of these engineered sequences to terminate transcription of the Escherichia coli RNA polymerase. Two out of three of the most successful T7 polymerase terminators also facilitated termination of the bacterial polymerase with around 99% efficiency.

Unique features of transcription termination and initiation at closely spaced tandem human genes

The synthesis of RNA Polymerase II (Pol2) products, which include messenger RNAs or long noncoding RNAs, culminates in transcription termination. How the transcriptional termination of a gene impacts the activity of promoters found immediately downstream of it, and which can be subject to potential transcriptional interference, remains largely unknown. We examined in an unbiased manner features of the intergenic region of pairs of tandem and closely spaced (<2kb) genes found on the same strand. Intergenic regions separating tandem genes are enriched with Guanines and are characterized by binding of several proteins, including AGO1 and AGO2 of the RNA interference pathway. Additionally, we found that Pol2 with a specific modification pattern is particularly enriched in this region, and it is lost upon perturbations affecting splicing or transcriptional elongation. Perturbations of genes involved in Pol2 pausing and R loop biology preferentially affect expression of downstream genes in tandem gene pairs. Overall, we find that features associated with potential Pol2 recycling rather than those associated with avoidance of transcriptional interference are the predominant driving force shaping these regions.

A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental gene partitioning in Toxoplasma

Correct 3'end processing of mRNAs is one of the regulatory cornerstones of gene expression. In a parasite that must adapt to the regulatory requirements of its multi-host life style, there is a need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly-arranged stage specific genes. In this study, we report our findings in T. gondii of an m6A-dependent 3'end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition was associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. Nanopore direct RNA sequencing shows the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.

ZC3H4 restricts non-coding transcription in human cells

The human genome encodes thousands of non-coding RNAs. Many of these terminate early and are then rapidly degraded, but how their transcription is restricted is poorly understood. In a screen for protein-coding gene transcriptional termination factors, we identified ZC3H4. Its depletion causes upregulation and extension of hundreds of unstable transcripts, particularly antisense RNAs and those transcribed from so-called super-enhancers. These loci are occupied by ZC3H4, suggesting that it directly functions in their transcription. Consistently, engineered tethering of ZC3H4 to reporter RNA promotes its degradation by the exosome. ZC3H4 is predominantly metazoan - interesting when considering its impact on enhancer RNAs that are less prominent in single-celled organisms. Finally, ZC3H4 loss causes a substantial reduction in cell proliferation, highlighting its overall importance. In summary, we identify ZC3H4 as playing an important role in restricting non-coding transcription in multi-cellular organisms.

The Role of Replication Clamp-Loader Protein HolC of Escherichia coli in Overcoming Replication/Transcription Conflicts

ABSTRACT In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the processivity clamp, and clamp loader complex. The holC gene encodes an accessory protein (known as χ) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability, although mutants show growth impairment, genetic instability, and sensitivity to DNA damaging agents. In this study, we isolate spontaneous suppressor mutants in a ΔholC strain and identify these by whole-genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, or alleles of sspA, encoding stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (β), and RpoC (β′) RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA, rpoC, and sspA. An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose that transcription complexes block replication in holC mutants and that Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity. IMPORTANCE Transcription elongation complexes present an impediment to DNA replication. We provide evidence that one component of the replication clamp loader complex, HolC, of Escherichia coli is required to overcome these blocks. This genetic study of transcription factor effects on holC growth defects implicates Rho-dependent transcriptional termination and DksA function as critical. It also implicates, for the first time, a role of SspA, stringent starvation protein, in avoidance or tolerance of replication/replication conflicts. We speculate that HolC helps avoid or resolve collisions between replication and transcription complexes, which become toxic in HolC’s absence.

A plant-like mechanism coupling m6A reading to polyadenylation safeguards transcriptome integrity and developmental genes partitioning in Toxoplasma

Correct 3’end processing of mRNAs is regarded as one of the regulatory cornerstones of gene expression. In a parasite that must answer to the high regulatory requirements of its multi-host life style, there is a great need to adopt additional means to partition the distinct transcriptional signatures of the closely and tandemly-arranged stage specific genes. In this study, we report on our findings in T. gondii of an m6A-dependent 3’end polyadenylation serving as a transcriptional barrier at these loci. We identify the core polyadenylation complex within T. gondii and establish CPSF4 as a reader for m6A-modified mRNAs, via a YTH domain within its C-terminus, a feature which is shared with plants. We bring evidence of the specificity of this interaction both biochemically, and by determining the crystal structure at high resolution of the T. gondii CPSF4-YTH in complex with an m6A modified RNA. We show that the loss of m6A, both at the level of its deposition or its recognition was associated with an increase in aberrantly elongated chimeric mRNAs emanating from impaired transcriptional termination, a phenotype previously noticed in the plant model Arabidopsis thaliana. We bring Nanopore direct RNA sequencing-based evidence of the occurrence of transcriptional read-through breaching into downstream repressed stage-specific genes, in the absence of either CPSF4 or the m6A RNA methylase components in both T. gondii and A. thaliana. Taken together, our results shed light on an essential regulatory mechanism coupling the pathways of m6A metabolism directly to the cleavage and polyadenylation processes, one that interestingly seem to serve, in both T. gondii and A. thaliana, as a guardian against aberrant transcriptional read-throughs.Highlightsm6A is recognized in apicomplexan and plants by CPSF4, a member of the cleavage and polyadenylation complex machinery.The structural insight behind the specificity of the binding of m6A by the CPSF4 YTH subunit are solved by high resolution crystal structures.The m6A-driven 3’end polyadenylation pathway protects transcriptome integrity by restricting transcriptional read-throughs and RNA chimera formation in apicomplexan parasites and plants.

The role of replication clamp-loader protein HolC of Escherichia coli in overcoming replication / transcription conflicts

ABSTRACTIn Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits as well as the processivity clamp and clamp loader complex. The holC gene encodes an accessory protein (known as x) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability although mutants show growth impairment, genetic instability and sensitivity to DNA damaging agents. In this study, to elucidate the role of HolC in replication, we isolate spontaneous suppressor mutants in a holCΔ strain and identify these by whole genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains or sspA, stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (β) and RpoC (β’) RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. Mfd had no effect nor did elongation factors GreA and GreB. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA rpoC and sspA.These results are consistent with the hypothesis that transcription complexes block replication in holC mutants and Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity.IMPORTANCETranscription elongation complexes present an impediment to DNA replication. We provide evidence that one component of the replication clamp loader complex, HolC, of E. coli is required to overcome these blocks. This genetic study of transcription factor effects on holC growth defects implicates Rho-dependent transcriptional termination and DksA function as critical. It also implicates, for the first time, a role of SspA, stringent starvation protein, in avoidance or tolerance of replication/replication conflicts. We speculate that HolC helps resolve codirectional collisions between replication and transcription complexes, which become toxic in HolC’s absence.