UV-induced inhibition of transcription involves repression of transcription initiation and phosphorylation of RNA polymerase II

Cells from patients with Cockayne syndrome (CS) are hypersensitive to DNA-damaging agents and are unable to restore damage-inhibited RNA synthesis. On the basis of repair kinetics of different types of lesions in transcriptionally active genes, we hypothesized previously that impaired transcription in CS cells is a consequence of defective transcription initiation after DNA damage induction. Here, we investigated the effect of UV irradiation on transcription by using an in vitro transcription system that allowed uncoupling of initiation from elongation events. Nuclear extracts prepared from UV-irradiated or mock-treated normal human and CS cells were assayed for transcription activity on an undamaged β-globin template. Transcription activity in nuclear extracts closely mimicked kinetics of transcription in intact cells: extracts from normal cells prepared 1 h after UV exposure showed a strongly reduced activity, whereas transcription activity was fully restored in extracts prepared 6 h after treatment. Extracts from CS cells exhibited reduced transcription activity at any time after UV exposure. Reduced transcription activity in extracts coincided with a strong reduction of RNA polymerase II (RNAPII) containing hypophosphorylated C-terminal domain, the form of RNAPII known to be recruited to the initiation complex. These results suggest that inhibition of transcription after UV irradiation is at least partially caused by repression of transcription initiation and not solely by blocked elongation at sites of lesions. Generation of hypophosphorylated RNAPII after DNA damage appears to play a crucial role in restoration of transcription. CS proteins may be required for this process in a yet unknown way.

An initiation element in the yeast CUP1 promoter is recognized by RNA polymerase II in the absence of TATA box-binding protein if the DNA is negatively supercoiled

Purified RNA polymerase II initiated transcription from the yeast CUP1 promoter fused to a C-less cassette if the DNA was negatively supercoiled. Relaxed plasmid was not transcribed. Transcription did not require addition of any other transcription factors. TATA box-binding protein (TBP) was not detectable in the polymerase preparation and the TATA box was not required. Deletion analysis of the CUP1 promoter revealed that a 25-bp element containing the initiation region was sufficient for recognition by polymerase. Two transcription start sites were mapped, one of which is identical to one of the two major start sites observed in vivo. Our observations can be accounted for by using a theoretical analysis of the probability of DNA melting within the plasmid as a function of superhelix density: the CUP1 initiation element is intrinsically unstable to superhelical stress, permitting entry of the polymerase, which then scans the DNA to locate the start site. In support of this analysis, the CUP1 promoter was sensitive to mung bean nuclease. These observations and a previous theoretical analysis of yeast genes support the idea that promoters are stress points within the DNA superhelix. The role of transcription factors might be to mark the promoter and to regulate specific melting of promoter DNA.

Crystal structure of yeast initiation factor 4A, a DEAD-box RNA helicase

The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D/H)-box RNA helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 Å resolution; it has a parallel α-β topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related helicases. Using data to 2.8 Å resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a “dumbbell” structure consisting of two compact domains connected by an extended linker. By using the structures of other helicases as a template, compact structures can be modeled for eIF4A that suggest (i) helicase motif IV binds RNA; (ii) Arg-298, which is conserved in the DEA(D/H)-box RNA helicase family but is absent from many other helicases, also binds RNA; and (iii) motifs V and VI “link” the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D/H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding.

ROP-1, an RNA quality-control pathway component, affects Caenorhabditis elegans dauer formation

Caenorhabditis elegans dauer formation is an alternative larval developmental pathway that the worm can take when environmental conditions become detrimental. Animals can survive several months in this stress-resistant stage and can resume normal development when growth conditions improve. Although the worms integrate a variety of sensory information to commit to dauer formation, it is currently unknown whether they also monitor internal cellular damage. The Ro ribonucleoprotein complex, which was initially described as a human autoantigen, is composed of one major 60-kDa protein, Ro60, that binds to one of four small RNA molecules, designated Y RNAs. Ro60 has been shown to bind mutant 5S rRNA molecules in Xenopus oocytes, suggesting a role for Ro60 in 5S rRNA biogenesis. Analysis of ribosomes from a C. elegans rop-1(−) strain, which is null for the expression of Ro60, demonstrated that they contain a high percentage of mutant 5S rRNA molecules, thereby strengthening the notion of a link between the rop-1 gene product and 5S rRNA quality control. The Ro particle was recently shown to be involved in the resistance of Deinococcus radiodurans to UV irradiation, suggesting a role for the Ro complex in stress resistance. We have studied the role of rop-1 in dauer formation. We present genetic and biochemical evidence that rop-1 interacts with dauer-formation genes and is involved in the regulation of the worms' entry into the dauer stage. Furthermore, we find that the rop-1 gene product undergoes a proteolytic processing step that is regulated by the dauer formation pathway via an aspartic proteinase. These results suggest that the Ro particle may function in an RNA quality-control checkpoint for dauer formation.

Mapping the σ70 subunit contact sites on Escherichia coli RNA polymerase with a σ70-conjugated chemical protease

The core enzyme of Escherichia coli RNA polymerase acquires essential promoter recognition and transcription initiation activities by binding one of several σ subunits. To characterize the proximity between σ70, the major σ for transcription of the growth-related genes, and the core enzyme subunits (α2ββ′), we analyzed the protein-cutting patterns produced by a set of covalently tethered FeEDTA probes [FeBABE: Fe (S)-1-(p-bromoacetamidobenzyl)EDTA]. The probes were positioned in or near conserved regions of σ70 by using seven mutants, each carrying a single cysteine residue at position 132, 376, 396, 422, 496, 517, or 581. Each FeBABE-conjugated σ70 was bound to the core enzyme, which led to cleavage of nearby sites on the β and β′ subunits (but not α). Unlike the results of random cleavage [Greiner, D. P., Hughes, K. A., Gunasekera, A. H. & Meares, C. F. (1996) Proc. Natl. Acad. Sci. USA 93, 71–75], the cut sites from different probe-modified σ70 proteins are clustered in distinct regions of the subunits. On the β subunit, cleavage is observed in two regions, one between residues 383 and 554, including the conserved C and Rif regions; and the other between 854 and 1022, including conserved region G, regions of ppGpp sensitivity, and one of the segments forming the catalytic center of RNA polymerase. On the β′ subunit, the cleavage was identified within the sequence 228–461, including β′ conserved regions C and D (which comprise part of the catalytic center).

Translation inhibitors stabilize Escherichia coli mRNAs independently of ribosome protection

Translation inhibitors such as chloramphenicol in prokaryotes or cycloheximide in eukaryotes stabilize many or most cellular mRNAs. In Escherichia coli, this stabilization is ascribed generally to the shielding of mRNAs by stalled ribosomes. To evaluate this interpretation, we examine here how inhibitors affect the stabilities of two untranslated RNAs, i.e., an engineered lacZ mRNA lacking a ribosome binding site, and a small regulatory RNA, RNAI. Whether they block elongation or initiation, all translation inhibitors tested stabilized these RNAs, indicating that stabilization does not necessarily reflect changes in packing or activity of translating ribosomes. Moreover, both the initial RNase E-dependent cleavage of RNAI and lacZ mRNA and the subsequent attack of RNAI by polynucleotide phosphorylase and poly(A)-polymerase were slowed. Among various possible mechanisms for this stabilization, we discuss in particular a passive model. When translation is blocked, rRNA synthesis is known to increase severalfold and rRNA becomes unstable. Meanwhile, the pools of RNase E and polynucleotide phosphorylase, which, in growing cells, are limited because these RNases autoregulate their own synthesis, cannot expand. The processing/degradation of newly synthesized rRNA would then titrate these RNases, causing bulk mRNA stabilization.

A trans-acting RNA as a control switch in Escherichia coli: DsrA modulates function by forming alternative structures

DsrA is an 87-nucleotide regulatory RNA of Escherichia coli that acts in trans by RNA–RNA interactions with two different mRNAs, hns and rpoS. DsrA has opposite effects on these transcriptional regulators. H-NS levels decrease, whereas RpoS (σs) levels increase. Here we show that DsrA enhances hns mRNA turnover yet stabilizes rpoS mRNA, either directly or via effects on translation. Computational and RNA footprinting approaches led to a refined structure for DsrA, and a model in which DsrA interacts with the hns mRNA start and stop codon regions to form a coaxial stack. Analogous bipartite interactions exist in eukaryotes, albeit with different regulatory consequences. In contrast, DsrA base pairs in discrete fashion with the rpoS RNA translational operator. Thus, different structural configurations for DsrA lead to opposite regulatory consequences for target RNAs.

A double-stranded RNA element from a hypovirulent strain of Rhizoctonia solani occurs in DNA form and is genetically related to the pentafunctional AROM protein of the shikimate pathway

M2 is a double-stranded RNA (dsRNA) element occurring in the hypovirulent isolate Rhs 1A1 of the plant pathogenic basidiomycete Rhizoctonia solani. Rhs 1A1 originated as a sector of the virulent field isolate Rhs 1AP, which contains no detectable amount of the M2 dsRNA. The complete sequence (3,570 bp) of the M2 dsRNA has been determined. A 6.9-kbp segment of total DNA from either Rhs 1A1 or Rhs 1AP hybridizes with an M2-specific cDNA probe. The sequences of M2 dsRNA and of PCR products generated from Rhs 1A1 total DNA were found to be identical. Thus this report describes a fungal host containing full-length DNA copies of a dsRNA element. A major portion of the M2 dsRNA is located in the cytoplasm, whereas a smaller amount is found in mitochondria. Based on either the universal or the mitochondrial genetic code of filamentous fungi, one strand of M2 encodes a putative protein of 754 amino acids. The resulting polypeptide has all four motifs of a dsRNA viral RNA-dependent RNA polymerase (RDRP) and is phylogenetically related to the RDRP of a mitochondrial dsRNA associated with hypovirulence in strain NB631 of Cryphonectria parasitica, incitant of chestnut blight. This polypeptide also has significant sequence similarity with two domains of a pentafunctional polypeptide, which catalyzes the five central steps of the shikimate pathway in yeast and filamentous fungi.

The 3′ untranslated region of a rice α-amylase gene functions as a sugar-dependent mRNA stability determinant

In plants, sugar feedback regulation provides a mechanism for control of carbohydrate allocation and utilization among tissues and organs. The sugar repression of α-amylase gene expression in rice provides an ideal model for studying the mechanism of sugar feedback regulation. We have shown previously that sugar repression of α-amylase gene expression in rice suspension cells involves control of both transcription rate and mRNA stability. The α-amylase mRNA is significantly more stable in sucrose-starved cells than in sucrose-provided cells. To elucidate the mechanism of sugar-dependent mRNA turnover, we have examined the effect of αAmy3 3′ untranslated region (UTR) on mRNA stability by functional analyses in transformed rice suspension cells. We found that the entire αAmy3 3′ UTR and two of its subdomains can independently mediate sugar-dependent repression of reporter mRNA accumulation. Analysis of reporter mRNA half-lives demonstrated that the entire αAmy3 3′ UTR and the two subdomains each functioned as a sugar-dependent destabilizing determinant in the turnover of mRNA. Nuclear run-on transcription analysis further confirmed that the αAmy3 3′ UTR and the two subdomains did not affect the transcription rate of promoter. The identification of sequence elements in the α-amylase mRNA that dictate the differential stability has very important implications for the study of sugar-dependent mRNA decay mechanisms.

Precisely full length, circularizable, complementary RNA: An infectious form of potato spindle tuber viroid

The replication of many viral and subviral pathogens as well as the amplification of certain cellular genes proceeds via a rolling circle mechanism. For potato spindle tuber (PSTVd) and related viroids, the possible role of a circular (−)strand RNA as a template for synthesis of (+)strand progeny is unclear. Infected plants appear to contain only multimeric linear (−)strand RNAs, and attempts to initiate infection with multimeric (−)PSTVd RNAs generally have failed. To examine critically the infectivity of monomeric (−)strand viroid RNAs, we have developed a ribozyme-based expression system for the production of precisely full length (−)strand RNAs whose termini are capable of undergoing facile circularization in vitro. Mechanical inoculation of tomato seedlings with electrophoretically purified (−)PSTVd RNA led to a small fraction of plants becoming infected whereas parallel assays with an analogous tomato planta macho viroid (−)RNA resulted in a much larger fraction of infected plants. Ribozyme-mediated production of (−)PSTVd RNA in transgenic plants led to the appearance of monomeric circular (−)PSTVd RNA and large amounts of (+)PSTVd progeny. No monomeric circular (−)PSTVd RNA could be detected in naturally infected plants by using either ribonuclease protection or electrophoresis under partially denaturing conditions. Although not a component of the normal replicative pathway, precisely full length (−)PSTVd RNA appears to contain all of the structural and regulatory elements necessary for initiation of viroid replication.

Resected RNA pseudoknots and their recognition by histidyl-tRNA synthetase

Duplexes constituted by closed or open RNA circles paired to single-stranded oligonucleotides terminating with 3′-CCAOH form resected pseudoknots that are substrates of yeast histidyl-tRNA synthetase. Design of this RNA fold is linked to the mimicry of the pseudoknotted amino acid accepting branch of the tRNA-like domain from brome mosaic virus, known to be charged by tyrosyl-tRNA synthetases, with RNA minihelices recapitulating accepting branches of canonical tRNAs. Prediction of the histidylation function of the new family of minimalist tRNA-like structures relates to the geometry of resected pseudoknots that allows proper presentation to histidyl-tRNA synthetase of analogues of the histidine identity determinants N-1 and N73 present in tRNAs. This geometry is such that the analogue of the major N-1 histidine determinant in the RNA circles faces the analogue of the discriminator N73 nucleotide in the accepting oligonucleotides. The combination of identity elements found in tRNAHis species from archaea, eubacteria, and organelles (G-1/C73) is the most efficient for determining histidylation of the duplexes. The inverse combination (C-1/G73) leads to the worst histidine acceptors with charging efficiencies reduced by 2–3 orders of magnitude. Altogether, these findings open new perspectives for understanding evolution of tRNA identity and serendipitous RNA functions.

Evolution of a quadripartite hybrid virus by interspecific exchange and recombination between replicase components of two related tripartite RNA viruses

Cucumber mosaic virus (CMV) and tomato aspermy virus (TAV) belong to the Cucumovirus genus. They have a tripartite genome consisting of single-stranded RNAs, designated 1, 2, and 3. Previous studies have shown that viable pseudorecombinants could be created in vitro by reciprocal exchanges between CMV and TAV RNA 3, but exchanges of RNAs 1 and 2 were replication deficient. When we coinoculated CMV RNAs 2 and 3 along with TAV RNAs 1 and 2 onto Nicotiana benthamiana, a hybrid quadripartite virus appeared that consisted of TAV RNA 1, CMV RNAs 2 and 3, and a distinctive chimeric RNA originating from a recombination between CMV RNA 2 and the 3′-terminal 320 nucleotides of TAV RNA 2. This hybrid arose by means of segment reassortment and RNA recombination to produce an interspecific hybrid with the TAV helicase subunit and the CMV polymerase subunit. To our knowledge, this is the first report demonstrating the evolution of a new plant or animal virus strain containing an interspecific hybrid replicase complex.

Formation of the preprimosome protects λ O from RNA transcription-dependent proteolysis by ClpP/ClpX

Using the bacteriophage λ DNA replication system, composed entirely of purified proteins, we have tested the accessibility of the short-lived λ O protein to the ClpP/ClpX protease during the various stages of λ DNA replication. We find that binding of λ O protein to its oriλ DNA sequence, leading to the so-called “O-some” formation, largely inhibits its degradation. On the contrary, under conditions permissive for transcription, the λ O protein bound to the oriλ sequence becomes largely accessible to ClpP/ClpX-mediated proteolysis. However, when the λ O protein is part of the larger oriλ:O⋅P⋅DnaB preprimosomal complex, transcription does not significantly increase ClpP/ClpX-dependent λ O degradation. These results show that transcription can stimulate proteolysis of a protein that is required for the initiation of DNA replication.

RNA polymerase subunit RPB5 plays a role in transcriptional activation

A mutation in RPB5 (rpb5–9), an essential RNA polymerase subunit assembled into RNA polymerases I, II, and III, revealed a role for this subunit in transcriptional activation. Activation by GAL4-VP16 was impaired upon in vitro transcription with mutant whole-cell extracts. In vivo experiments using inducible reporter plasmids and Northern analysis support the in vitro data and demonstrate that RPB5 influences activation at some, but not all, promoters. Remarkably, this mutation maps to a conserved region of human RPB5 implicated by others to play a role in activation. Chimeric human-yeast RPB5 containing this conserved region now can function in place of its yeast counterpart. The defects noted with rpb5–9 are similar to those seen in truncation mutants of the RPB1-carboxyl terminal domain (CTD). We demonstrate that RPB5 and the RPB1-CTD have overlapping roles in activation because the double mutant is synthetically lethal and has exacerbated activation defects at the GAL1/10 promoter. These studies demonstrate that there are multiple activation targets in RNA polymerase II and that RPB5 and the CTD have similar roles in activation.

HNS, a nuclear-cytoplasmic shuttling sequence in HuR

Proteins are transported into and out of the cell nucleus via specific signals. The two best-studied nuclear transport processes are mediated either by classical nuclear localization signals or nuclear export signals. There also are shuttling sequences that direct the bidirectional transport of RNA-binding proteins. Two examples are the M9 sequence in heterogeneous nuclear ribonucleoprotein A1 and the heterogeneous nuclear ribonucleoprotein K shuttling domain (KNS) sequence in heterogeneous nuclear ribonucleoprotein K, both of which appear to contribute importantly to the export of mRNA to the cytoplasm. HuR is an RNA-binding protein that can stabilize labile mRNAs containing AU-rich elements in their 3′ untranslated regions and has been shown to shuttle between the nucleus and cytoplasm (18, 19). We have identified in HuR a shuttling sequence that also possess transcription-dependent nuclear localization signal activity. We propose that HuR first may bind AU-rich element-containing mRNAs in the nucleus and then escort them through the nuclear pore, providing protection during and after export to the cytoplasmic compartment.