A nebulin super-repeat panel reveals stronger actin binding toward the ends of the super-repeat region.

INTRODUCTION: Nebulin is a giant actin-binding protein in the thin filament of the skeletal muscle sarcomere. Studies of nebulin interactions are limited by the size, complexity, and poor solubility of the protein. We divided the nebulin super-repeat region into a super-repeat panel, and studied nebulin/actin interactions. METHODS: Actin binding was studied using a co-sedimentation assay with filamentous actin and 26 different nebulin super-repeats. RESULTS: The panel revealed notable differences in actin binding between the super-repeats. Both ends of the super-repeat region bound actin significantly more strongly, whereas the central part of the protein bound actin weakly. Thus, the binding between nebulin and actin formed a location-dependent pattern of strong vs. weak binding. DISCUSSION: The nebulin super-repeat panel allowed us to study the actin binding of each super-repeat individually. The panel will be a powerful tool in elucidating nebulin function in health and disease. Muscle Nerve 59:116-121, 2019.

A Developmental Program Truncates Long Transcripts to Temporally Regulate Cell Signaling.

Rapid mitotic divisions and a fixed transcription rate limit the maximal length of transcripts in early Drosophila embryos. Previous studies suggested that transcription of long genes is initiated but aborted, as early nuclear divisions have short interphases. Here, we identify long genes that are expressed during short nuclear cycles as truncated transcripts. The RNA binding protein Sex-lethal physically associates with transcripts for these genes and is required to support early termination to specify shorter transcript isoforms in early embryos of both sexes. In addition, one truncated transcript for the gene short-gastrulation encodes a product in embryos that functionally relates to a previously characterized dominant-negative form, which maintains TGF-ß signaling in the off-state. In summary, our results reveal a developmental program of short transcripts functioning to help temporally regulate Drosophila embryonic development, keeping cell signaling at early stages to a minimum in order to support its proper initiation at cellularization.

Regulatory genes coordinating antibiotic-induced changes in promoter activity and early transcriptional termination of the mycobacterial intrinsic resistance gene whiB7.

Diseases caused by various Mycobacterium sp., especially Mycobacterium tuberculosis, are a major burden on global health care. Due to high intrinsic antibiotic resistance, treatment options are severely limited. In mycobacteria, WhiB7 coordinates intrinsic resistance to a broad range of antibiotics. While WhiB7 has been established as an auto-regulatory transcriptional activator, the signals and genes needed to induce its expression are poorly understood. Using Mycobacterium smegmatis as a model, we coupled transposon mutagenesis and next generation sequencing with WhiB7-specific antibiotic selection to identify genes that contribute to WhiB7 regulation and function. We showed that whiB7 expression was regulated by two coordinated processes: early termination of the whiB7 transcript and increased whiB7 promoter activity. Early termination was irreversibly maintained by constitutive expression of a putative aspartate aminotransferase gene, MSMEG_4060. A pair of hypothetical genes, MSMEG_3637 and MSMEG_3638, were identified as important contributors to whiB7 promoter induction on antibiotic challenge. Expansion of our understanding of the WhiB7-resistance pathway may lead to identification of inhibitors that allow the use of previously ineffective antibiotics to treat mycobacterial diseases.

Promoter-Terminator Gene Loops Affect Alternative 3'-End Processing in Yeast.

Many eukaryotic genes undergo alternative 3'-end poly(A)-site selection producing transcript isoforms with 3'-UTRs of different lengths and post-transcriptional fates. Gene loops are dynamic structures that juxtapose the 3'-ends of genes with their promoters. Several functions have been attributed to looping, including memory of recent transcriptional activity and polarity of transcription initiation. In this study, we investigated the relationship between gene loops and alternative poly(A)-site. Using the KlCYC1 gene of the yeast Kluyveromyces lactis, which includes a single promoter and two poly(A) sites separated by 394 nucleotides, we demonstrate in two yeast species the formation of alternative gene loops (L1 and L2) that juxtapose the KlCYC1 promoter with either proximal or distal 3'-end processing sites, resulting in the synthesis of short and long forms of KlCYC1 mRNA. Furthermore, synthesis of short and long mRNAs and formation of the L1 and L2 loops are growth phase-dependent. Chromatin immunoprecipitation experiments revealed that the Ssu72 RNA polymerase II carboxyl-terminal domain phosphatase, a critical determinant of looping, peaks in early log phase at the proximal poly(A) site, but as growth phase advances, it extends to the distal site. These results define a cause-and-effect relationship between gene loops and alternative poly(A) site selection that responds to different physiological signals manifested by RNA polymerase II carboxyl-terminal domain phosphorylation status.

Fail-safe transcriptional termination for protein-coding genes in S. cerevisiae.

Transcription termination of RNA polymerase II (Pol II) on protein-coding genes in S. cerevisiae relies on pA site recognition by 3' end processing factors. Here we demonstrate the existence of two alternative termination mechanisms that rescue polymerases failing to disengage from the template at pA sites. One of these fail-safe mechanisms is mediated by the NRD complex, similar to termination of short noncoding genes. The other termination mechanism is mediated by Rnt1 cleavage of the nascent transcript. Both fail-safe termination mechanisms trigger degradation of readthrough transcripts by the exosome. However, Rnt1-mediated termination can also enhance the usage of weak pA signals and thereby generate functional mRNA. We propose that these alternative Pol II termination pathways serve the dual function of avoiding transcription interference and promoting rapid removal of aberrant transcripts.

Yeast RNase III triggers polyadenylation-independent transcription termination.

Transcription termination of messenger RNA (mRNA) is normally achieved by polyadenylation followed by Rat1p-dependent 5'-3' exoribonuleolytic degradation of the downstream transcript. Here we show that the yeast ortholog of the dsRNA-specific ribonuclease III (Rnt1p) may trigger Rat1p-dependent termination of RNA transcripts that fail to terminate near polyadenylation signals. Rnt1p cleavage sites were found downstream of several genes, and the deletion of RNT1 resulted in transcription readthrough. Inactivation of Rat1p impaired Rnt1p-dependent termination and resulted in the accumulation of 3' end cleavage products. These results support a model for transcription termination in which cotranscriptional cleavage by Rnt1p provides access for exoribonucleases in the absence of polyadenylation signals.

The relaxing ori-ter balance of Mycoplasma genomes.

Mycoplasma are wall-less bacteria with small genomes, which are thought to have resulted from massive genome reductive processes, during which the ori-ter balance may be disrupted. For technical difficulties, ori and ter have been located only in a few Mycoplasma strains. Using the Z curve method, we were able to locate turning points on the Mycoplasma genomes, with the minimum and maximum points co-locating with ori or ter in the reference genomes. Assuming Z curve correctly located ori and ter, we calculated the distances from ori to ter in both directions on the circular genome and calculated the ori-ter balance status. The Mycoplasma genomes were not balanced, possibly as a result of close association of Mycoplasma with hosts, where there would be no other microbes for Mycoplasma to compete with for nutrients, so fastest possible growth related to balanced genomes might not be needed by Mycoplasma, leading to a relaxing ori-ter balance.

Systematic mutagenesis of the thymidine tract of the pyrBI attenuator and its effects on intrinsic transcription termination in Escherichia coli.

The pyrBI attenuator of Escherichia coli is an intrinsic transcription terminator composed of DNA with a hyphenated dyad symmetry and an adjacent 8 bp T:A tract (T-tract). These elements specify a G+C-rich terminator hairpin followed by a run of eight uridine residues (U-tract) in the RNA transcript. In this study, we examined the effects on in vivo transcription termination of systematic base substitutions in the T/U-tract of the pyrBI attenuator. We found that these substitutions diminished transcription termination efficiency to varying extents, depending on the nature and position of the substitution. In general, substitutions closer to the dyad symmetry/terminator hairpin exhibited the most significant effects. Additionally, we examined the effects on in vivo transcription termination of mutations that insert from 1 to 4 bases between the terminator hairpin and U-tract specified by the pyrBI attenuator. Our results show an inverse relationship between termination efficiency and the number of bases inserted. The effects of the substitution and insertion mutations on termination efficiency at the pyrBI attenuator were also measured in vitro, which corroborated the in vivo results. Our results are discussed in terms of the current models for intrinsic transcription termination and estimating termination efficiencies at intrinsic terminators of other bacteria.

Initiation of decay of Bacillus subtilis trp leader RNA.

Transcription termination in the leader region of the Bacillus subtilis trp operon is regulated by binding of the 11-mer TRAP complex to nascent trp RNA, which results in formation of a terminator structure. Rapid decay of trp leader RNA, which is required to release the TRAP complex and maintain a sufficient supply of free TRAP, is mediated by polynucleotide phosphorylase (PNPase). Using purified B. subtilis PNPase, we showed that, when TRAP was present, PNPase binding to the 3' end of trp leader RNA and PNPase digestion of trp leader RNA from the 3' end were inefficient. These results suggested that initiation of trp leader RNA may begin with an endonuclease cleavage upstream of the transcription terminator structure. Such cleavage was observed in vivo. Mutagenesis of nucleotides at the cleavage site abolished processing and resulted in a 4-fold increase in trp leader RNA half-life. This is the first mapping of a decay-initiating endonuclease cleavage site on a native B. subtilis RNA.

Insertion of group II intron retroelements after intrinsic transcriptional terminators.

Mobile DNAs use many mechanisms to minimize damage to their hosts. Here we show that a subclass of group II introns avoids host damage by inserting directly after transcriptional terminator motifs in bacterial genomes (stem-loops followed by Ts). This property contrasts with the site-specific behavior of most group II introns, which insert into homing site sequences. Reconstituted ribonucleo protein particles of the Bacillus halodurans intron B.h.I1 are shown to reverse-splice into DNA targets in vitro but require the DNA to be single-stranded and fold into a stem-loop analogous to the RNA structure that forms during transcription termination. Recognition of this DNA stem-loop motif accounts for in vivo target specificity. Insertion after terminators is a previously unrecognized strategy for a selfish DNA because it prevents interruption of coding sequences and restricts expression of the mobile DNA after integration.

How Rho exerts its muscle on RNA.

Rho factor in bacteria terminates transcription by using energy from ATP hydrolysis to forcefully dissociate the transcripts from RNA polymerase. used data from presteady-state ATPase kinetics to support a rational mechanistic model for Rho's action on RNA.

New construct approaches for efficient gene silencing in plants.

An important component of conventional sense, antisense, and double-strand RNA-based gene silencing constructs is the transcriptional terminator. Here, we show that this regulatory element becomes obsolete when gene fragments are positioned between two oppositely oriented and functionally active promoters. The resulting convergent transcription triggers gene silencing that is at least as effective as unidirectional promoter-to-terminator transcription. In addition to short, variably sized, and nonpolyadenylated RNAs, terminator-free cassette produced rare, longer transcripts that reach into the flanking promoter. These read-through products did not influence the efficacy and expression levels of the neighboring hygromycin phosphotransferase gene. Replacement of gene fragments by promoter-derived sequences further increased the extent of gene silencing. This finding indicates that genomic DNA may be a more efficient target for gene silencing than gene transcripts.

Regulation of yeast NRD1 expression by premature transcription termination.

The yeast RNA binding proteins Nrd1 and Nab3 are required for termination of nonpolyadenylated transcripts from RNA polymerase (Pol) II-transcribed snRNA and snoRNA genes. In this paper, we show that NRD1 expression is regulated by Nrd1- and Nab3-directed premature termination. Sequences recognized by these proteins are present in NRD1 mRNA and are required for regulated expression. Chromatin immunoprecipitation and transcription run-on experiments show that, in wild-type cells, Pol II occupancy is high at the 5' end of the NRD1 gene and decreases at the 3' end. Mutation of Nrd1 and Nab3 binding sites within the NRD1 mRNA leads to a relative increase in Pol II occupancy of downstream sequences. We further show that NRD1 autoregulation involves components of the exosome and a newly discovered exosome-activating complex. Together, these results show that NRD1 is a eukaryotic cellular gene regulated through premature transcription termination.

Structural basis of HutP-mediated transcription anti-termination.

Bacteria often use anti-terminator proteins to sense a specific metabolite signal and direct RNA polymerase to either terminate transcription or transcribe the downstream genes of an operon. Although many proteins that regulate various operons using this mechanism have been identified, insights into their activation processes before cognate mRNA binding have remained obscure. HutP from Bacillus subtilis regulates the hut operon by an anti-termination mechanism. Recently, several crystal structures of HutP [apo-HutP, HutP-L-histidine (and histidine analog), HutP-L-histidine-Mg(2+) and HutP-L-histidine-Mg(2+)-RNA] have been reported. These structural and functional studies of HutP have revealed how the protein undergoes conformational changes in response to two key components: L-histidine and Mg(2+) ions.

A role for the CPF 3'-end processing machinery in RNAP II-dependent gene looping.

The prevailing view of the RNA polymerase II (RNAP II) transcription cycle is that RNAP II is recruited to the promoter, transcribes a linear DNA template, then terminates transcription and dissociates from the template. Subsequent rounds of transcription are thought to require de novo recruitment of RNAP II to the promoter. Several recent findings, including physical interaction of 3'-end processing factors with both promoter and terminator regions, challenge this concept. Here we report a physical association of promoter and terminator regions of the yeast BUD3 and SEN1 genes. These interactions are transcription-dependent, require the Ssu72 and Pta1 components of the CPF 3'-end processing complex, and require the phosphatase activity of Ssu72. We propose a model for RNAP II transcription in which promoter and terminator regions are juxtaposed, and that the resulting gene loops facilitate transcription reinitiation by the same molecule of RNAP II in a manner dependent upon Ssu72-mediated CTD dephosphorylation.

Termination factor-mediated DNA loop between termination and initiation sites drives mitochondrial rRNA synthesis.

The human mitochondrial transcription termination factor mTERF plays a central role in the control of heavy-strand rDNA transcription by promoting initiation, besides termination, of this transcription. However, until now, the mechanism underlying this stimulation of transcription by mTERF was not understood. In the present work, addition of mTERF to a HeLa cell mitochondrial lysate-based reaction mixture containing an artificial rDNA template did indeed specifically stimulate rDNA transcription. This stimulation required that mTERF be simultaneously bound to the rDNA transcription termination and initiation sites in the same molecule, thus forming a loop. Most significantly, a double binding of mTERF to the rDNA molecule, with resulting loop formation, was also shown in vivo. These results strongly suggest that, to satisfy the need for high rate of rDNA transcription, human mitochondrial rRNA synthesis involves mTERF-mediated rDNA looping that promotes recycling of the transcription machinery.

A quantitative description of the binding states and in vitro function of antitermination protein N of bacteriophage lambda.

The N protein of bacteriophage lambda activates transcription of genes that lie downstream of termination sequences by suppressing transcription termination. N binds to specific (boxB) and non-specific sites on the transcript RNA and contacts RNA polymerase via cis-RNA looping, resulting in "antitermination" of transcription. To find the effect of N-boxB binding on antitermination, we quantitatively relate binding measurements made in isolation to in vitro antitermination activity. We measure binding of N to boxB RNA, non-specific single-stranded RNA, and non-specific double-stranded DNA fluorimetrically, and use an equilibrium model to describe quantitatively the binding of N to nucleic acids of Escherichia coli transcription elongation complexes. We then test the model by comparison with in vitro N antitermination activity measured in reactions containing these same elongation complexes. We find that binding of N protein to the nucleic acid components of transcription elongation complexes can quantitatively predict antitermination activity, suggesting that antitermination in vitro is determined by a nucleic acid binding equilibrium with one molecule of N protein per RNA transcript being sufficient for antitermination. Elongation complexes contain numerous overlapping non-specific RNA and DNA-binding sites for N; the large number of sites compensates for the low N binding affinity, so multiple N proteins are expected to bind to elongation complexes. The occupancy/activity of these proteins is described by a binomial distribution of proteins on transcripts containing multiple non-specific sites. The contribution of specific (boxB) binding to activity also depends on this distribution. Specificity is not measured accurately by measurements made in the presence and in the absence of boxB. We find that antitermination is inhibited by non-productive binding of N to non-specific sites on template DNA, and that NusA protein covers RNA sites on the transcript, limiting N access and activity. The activity and specificity of regulatory proteins that loop from high-affinity binding sites are likely modulated by multiple non-specific binding events; in vivo activity may also be regulated by the modulation of non-specific binding.

Genetic reprogramming by retroviruses: enhanced suppression of translational termination.

Viruses often exploit or subvert host machinery for their own purposes during replication. A search for proteins interacting with the murine leukemia virus reverse transcriptase (RT) recently provided a new example of such exploitation. RT was found to bind the eukaryotic translational release factor 1 (eRF1), the protein that recognizes stop codons and, in complex with eRF3, causes termination and polypeptide release from the ribosome. RT is derived from a large Gag-Pol polyprotein, and its synthesis requires a translational readthrough, a suppression of termination, at a stop codon at the end of the gag gene. The binding of eRF1 by RT was found to inhibit eRF1 action, enhance the efficiency of readthrough, and thus cause higher levels of RT synthesis. The observations suggest that retroviruses manipulate the translational machinery in sophisticated ways to fine-tune their own gene expression.

A Rho-dependent phase-variable transcription terminator controls expression of the FimE recombinase in Escherichia coli.

The fim switch is a 314 bp segment of invertible chromosomal DNA that is responsible for phase-variable expression of type 1 fimbriae in Escherichia coli. The switch harbours the promoter of the fimA gene. This codes for the type 1 fimbrial subunit protein and, when the promoter is directed towards fimA (phase ON), the bacteria are fimbriate and, when it is directed away, the cells are afimbriate. The switch lies immediately downstream from the fimE gene, coding for a tyrosine site-specific recombinase that catalyses inversion of the switch from the ON to the OFF phase. It has been suggested previously that, because the fim switch lies immediately downstream from the fimE gene, expression of FimE could be subject to control by antisense RNA in phase OFF bacteria in which the promoter harboured within the fim switch is oriented against the direction of transcription of the fimE gene. In this study, no effect of inducible fimE antisense RNA, expressed in cis or in trans, on FimE expression was detected. In phase ON cells, fimE mRNA extends across the switch into fimA, whereas in phase OFF cells, it terminates within the switch. This termination is Rho dependent and is abolished in a rho mutant. The extended fimE found in phase ON cells is more stable and results in an approximately fivefold increase in FimE protein compared with phase OFF bacteria. In the absence of Rho factor, fimE mRNA is equally stable in phase ON and phase OFF cells, and the levels of FimE recombinase are also equal.

Conserved economics of transcription termination in eubacteria.

A secondary structure in the nascent RNA followed by a trail of U residues is believed to be necessary and sufficient to terminate transcription. Such structures represent an extremely economical mechanism of transcription termination since they function in the absence of any additional protein factors. We have developed a new algorithm, GeSTer, to identify putative terminators and analysed all available complete bacterial genomes. The algorithm classifies the structures into five classes. We find that potential secondary structure sequences are concentrated downstream of coding regions in most bacterial genomes. Interestingly, many of these structures are not followed by a discernible U-trail. However, irrespective of the nature of the trail sequence, the structures show a similar distribution, indicating that they serve the same purpose. In contrast, such a distribution is absent in archaeal genomes, indicating that they employ a distinct mechanism for transcription termination. The present algorithm represents the fastest and most accurate algorithm for identifying terminators in eubacterial genomes without being restricted by the classical Escherichia coli paradigm.