Publications

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A
Gailus-Durner, V, Chintamaneni C, Wilson R, Brill SJ, Vershon AK.  1997.  Analysis of a Meiosis-specific uRS1 Site: Sequence Requirements and Involvement of Replication Protein a. Mol Cell Biol. 17:3536-3546. Abstract
URS1 is a transcriptional repressor site found in the promoters of a wide variety of yeast genes that are induced under stress conditions. In the context of meiotic promoters, URS1 sites act as repressor sequences during mitosis and function as activator sites during meiosis. We have investigated the sequence requirements of the URS1 site of the meiosis-specific HOP1 gene (URS1H) and have found differences compared with a URS1 site from a nonmeiotic gene. We have also observed that the sequence specificity for meiotic activation at this site differs from that for mitotic repression. Base pairs flanking the conserved core sequence enhance meiotic induction but are not required for mitotic repression of HOP1. Electrophoretic mobility shift assays of mitotic and meiotic cell extracts show a complex pattern of DNA-protein complexes, suggesting that several different protein factors bind specifically to the site. We have determined that one of the complexes of URS1H is formed by replication protein A (RPA). Although RPA binds to the double-stranded URS1H site in vitro, it has much higher affinity for single-stranded than for double-stranded URS1H, and one-hybrid assays suggest that RPA does not bind to this site at detectable levels in vivo. In addition, conditional-lethal mutations in RPA were found to have no effect on URS1H-mediated repression. These results suggest that although RPA binds to URS1H in vitro, it does not appear to have a functional role in transcriptional repression through this site in vivo.
Wang, W, Messing J.  2012.  Analysis of ADP-glucose pyrophosphorylase expression during turion formation induced by abscisic acid in Spirodela polyrhiza (greater duckweed). BMC Plant Biol. 12:5. AbstractWebsite
BACKGROUND: Aquatic plants differ in their development from terrestrial plants in their morphology and physiology, but little is known about the molecular basis of the major phases of their life cycle. Interestingly, in place of seeds of terrestrial plants their dormant phase is represented by turions, which circumvents sexual reproduction. However, like seeds turions provide energy storage for starting the next growing season. RESULTS: To begin a characterization of the transition from the growth to the dormant phase we used abscisic acid (ABA), a plant hormone, to induce controlled turion formation in Spirodela polyrhiza and investigated their differentiation from fronds, representing their growth phase, into turions with respect to morphological, ultra-structural characteristics, and starch content. Turions were rich in anthocyanin pigmentation and had a density that submerged them to the bottom of liquid medium. Transmission electron microscopy (TEM) of turions showed in comparison to fronds shrunken vacuoles, smaller intercellular space, and abundant starch granules surrounded by thylakoid membranes. Turions accumulated more than 60% starch in dry mass after two weeks of ABA treatment. To further understand the mechanism of the developmental switch from fronds to turions, we cloned and sequenced the genes of three large-subunit ADP-glucose pyrophosphorylases (APLs). All three putative protein and exon sequences were conserved, but the corresponding genomic sequences were extremely variable mainly due to the invasion of miniature inverted-repeat transposable elements (MITEs) into introns. A molecular three-dimensional model of the SpAPLs was consistent with their regulatory mechanism in the interaction with the substrate (ATP) and allosteric activator (3-PGA) to permit conformational changes of its structure. Gene expression analysis revealed that each gene was associated with distinct temporal expression during turion formation. APL2 and APL3 were highly expressed in earlier stages of turion development, while APL1 expression was reduced throughout turion development. CONCLUSIONS: These results suggest that the differential expression of APLs could be used to enhance energy flow from photosynthesis to storage of carbon in aquatic plants, making duckweeds a useful alternative biofuel feedstock.
Wang, W, Messing J.  2012.  Analysis of ADP-glucose pyrophosphorylase expression during turion formation induced by abscisic acid in Spirodela polyrhiza (greater duckweed). BMC Plant Biology. 12(5) Abstract
Aquatic plants differ in their development from terrestrial plants in their morphology and physiology, but little is known about the molecular basis of the major phases of their life cycle. Interestingly, in place of seeds of terrestrial plants their dormant phase is represented by turions, which circumvents sexual reproduction. However, like seeds turions provide energy storage for starting the next growing season.
Dong, J, Feng Y, Kumar D, Zhang W, Zhu T, Luo M-C, Messing J.  2016.  Analysis of tandem gene copies in maize chromosomal regions reconstructed from long sequence reads.. Proceedings of the National Academy of Sciences of the United States of America. 113(29):7949-56. Abstract
Haplotype variation not only involves SNPs but also insertions and deletions, in particular gene copy number variations. However, comparisons of individual genomes have been difficult because traditional sequencing methods give too short reads to unambiguously reconstruct chromosomal regions containing repetitive DNA sequences. An example of such a case is the protein gene family in maize that acts as a sink for reduced nitrogen in the seed. Previously, 41-48 gene copies of the alpha zein gene family that spread over six loci spanning between 30- and 500-kb chromosomal regions have been described in two Iowa Stiff Stalk (SS) inbreds. Analyses of those regions were possible because of overlapping BAC clones, generated by an expensive and labor-intensive approach. Here we used single-molecule real-time (Pacific Biosciences) shotgun sequencing to assemble the six chromosomal regions from the Non-Stiff Stalk maize inbred W22 from a single DNA sequence dataset. To validate the reconstructed regions, we developed an optical map (BioNano genome map; BioNano Genomics) of W22 and found agreement between the two datasets. Using the sequences of full-length cDNAs from W22, we found that the error rate of PacBio sequencing seemed to be less than 0.1% after autocorrection and assembly. Expressed genes, some with premature stop codons, are interspersed with nonexpressed genes, giving rise to genotype-specific expression differences. Alignment of these regions with those from the previous analyzed regions of SS lines exhibits in part dramatic differences between these two heterotic groups.
Hanlon, SE, Xu Z, Norris DN, Vershon AK.  2004.  Analysis of the Meiotic role of the Mitochondrial Ribosomal Proteins Mrps17 and Mrpl37 in Saccharomyces Cerevisiae. Yeast. 21:1241-1252. Abstract
Sporulation in the yeast Saccharomyces cerevisiae is a complex and tightly regulated pathway that involves the induction of a large number of genes. We have identified MRPS17 in a cDNA library enriched for sporulation-specific genes. Homology searches show that the first one-third of Mrps17 has strong sequence similarity to bacterial S17 proteins, suggesting that Mrps17 is a potential mitochondrial ribosomal protein. This is further supported by the fact that mrps17Delta cells are respiratory-deficient and that a Mrps17-GFP fusion localizes to the mitochondria. We have confirmed by Northern blot analysis that both MRPS17 and MRPL37 are strongly induced during the middle stages of sporulation and that this induction is dependent on the presence of a middle sporulation element (MSE) in the promoters of these genes. Interestingly, we found that Mrps17 and Mrpl37, but not other mitochondrial ribosomal proteins, accumulate during the middle stages of sporulation. These results suggest that Mrps17 and Mrpl37 may have additional meiosis-specific roles.
Murat, F, Xu JH, Tannier E, Abrouk M, Guilhot N, Pont C, Messing J, Salse J.  2010.  Ancestral grass karyotype reconstruction unravels new mechanisms of genome shuffling as a source of plant evolution. Genome Res. 20:1545-57. AbstractWebsite
The comparison of the chromosome numbers of today's species with common reconstructed paleo-ancestors has led to intense speculation of how chromosomes have been rearranged over time in mammals. However, similar studies in plants with respect to genome evolution as well as molecular mechanisms leading to mosaic synteny blocks have been lacking due to relevant examples of evolutionary zooms from genomic sequences. Such studies require genomes of species that belong to the same family but are diverged to fall into different subfamilies. Our most important crops belong to the family of the grasses, where a number of genomes have now been sequenced. Based on detailed paleogenomics, using inference from n = 5-12 grass ancestral karyotypes (AGKs) in terms of gene content and order, we delineated sequence intervals comprising a complete set of junction break points of orthologous regions from rice, maize, sorghum, and Brachypodium genomes, representing three different subfamilies and different polyploidization events. By focusing on these sequence intervals, we could show that the chromosome number variation/reduction from the n = 12 common paleo-ancestor was driven by nonrandom centric double-strand break repair events. It appeared that the centromeric/telomeric illegitimate recombination between nonhomologous chromosomes led to nested chromosome fusions (NCFs) and synteny break points (SBPs). When intervals comprising NCFs were compared in their structure, we concluded that SBPs (1) were meiotic recombination hotspots, (2) corresponded to high sequence turnover loci through repeat invasion, and (3) might be considered as hotspots of evolutionary novelty that could act as a reservoir for producing adaptive phenotypes.
Maffioli, SI, Zhang Y, Degen D, Carzaniga T, Del Gatto G, Serina S, Monciardini P, Mazzetti C, Guglierame P, Candiani G et al..  2017.  Antibacterial Nucleoside-Analog Inhibitor of Bacterial RNA Polymerase.. Cell. 169(7):1240-1248.e23. Abstract
Drug-resistant bacterial pathogens pose an urgent public-health crisis. Here, we report the discovery, from microbial-extract screening, of a nucleoside-analog inhibitor that inhibits bacterial RNA polymerase (RNAP) and exhibits antibacterial activity against drug-resistant bacterial pathogens: pseudouridimycin (PUM). PUM is a natural product comprising a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 6'-amino-pseudouridine. PUM potently and selectively inhibits bacterial RNAP in vitro, inhibits bacterial growth in culture, and clears infection in a mouse model of Streptococcus pyogenes peritonitis. PUM inhibits RNAP through a binding site on RNAP (the NTP addition site) and mechanism (competition with UTP for occupancy of the NTP addition site) that differ from those of the RNAP inhibitor and current antibacterial drug rifampin (Rif). PUM exhibits additive antibacterial activity when co-administered with Rif, exhibits no cross-resistance with Rif, and exhibits a spontaneous resistance rate an order-of-magnitude lower than that of Rif. PUM is a highly promising lead for antibacterial therapy.
Mukhopadhyay, J, Sineva E, Knight J, Levy RM, Ebright RH.  2004.  Antibacterial peptide microcin J25 inhibits transcription by binding within and obstructing the RNA polymerase secondary channel.. Molecular cell. 14(6):739-51. Abstract
The antibacterial peptide microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). Biochemical results indicate that inhibition of transcription occurs at the level of NTP uptake or NTP binding by RNAP. Genetic results indicate that inhibition of transcription requires an extensive determinant, comprising more than 50 amino acid residues, within the RNAP secondary channel (also known as the "NTP-uptake channel" or "pore"). Biophysical results indicate that inhibition of transcription involves binding of MccJ25 within the RNAP secondary channel. Molecular modeling indicates that binding of MccJ25 within the RNAP secondary channel obstructs the RNAP secondary channel. We conclude that MccJ25 inhibits transcription by binding within and obstructing the RNAP secondary channel--acting essentially as a "cork in a bottle." Obstruction of the RNAP secondary channel represents an attractive target for drug discovery.
Kuznedelov, K, Semenova E, Knappe T, Marahiel M, Ebright RHE, Severinov K.  2011.  Antibacterial threaded lasso-peptide capistruin is a DNA-dependent RNA polymerase inhibitor. J. Mol. Biol. 412:842-848.
Kuznedelov, K, Semenova E, Knappe TA, Mukhamedyarov D, Srivastava A, Chatterjee S, Ebright RH, Marahiel MA, Severinov K.  2011.  The Antibacterial Threaded-lasso Peptide Capistruin Inhibits Bacterial RNA Polymerase.. Journal of molecular biology. 412(5):842-8. Abstract
Capistruin, a ribosomally synthesized, post-translationally modified peptide produced by Burkholderia thailandensis E264, efficiently inhibits growth of Burkholderia and closely related Pseudomonas strains. The functional target of capistruin is not known. Capistruin is a threaded-lasso peptide (lariat peptide) consisting of an N-terminal ring of nine amino acids and a C-terminal tail of 10 amino acids threaded through the ring. The structure of capistruin is similar to that of microcin J25 (MccJ25), a threaded-lasso antibacterial peptide that is produced by some strains of Escherichia coli and targets DNA-dependent RNA polymerase (RNAP). Here, we show that capistruin, like MccJ25, inhibits wild type E. coli RNAP but not mutant, MccJ25-resistant, E. coli RNAP. We show further that an E. coli strain resistant to MccJ25, as a result of a mutation in an RNAP subunit gene, exhibits resistance to capistruin. The results indicate that the structural similarity of capistruin and MccJ25 reflects functional similarity and suggest that the functional target of capistruin, and possibly other threaded-lasso peptides, is bacterial RNAP.
Xiao, Y, Wei X, Ebright R, Wall D.  2011.  Antibiotic production by myxobacteria plays a role in predation.. Journal of bacteriology. 193(18):4626-33. Abstract
Myxobacteria are predatory and are prolific producers of secondary metabolites. Here, we tested a hypothesized role that secondary metabolite antibiotics function as weapons in predation. To test this, a Myxococcus xanthus Δta1 mutant, blocked in antibiotic TA (myxovirescin) production, was constructed. This TA(-) mutant was defective in producing a zone of inhibition (ZOI) against Escherichia coli. This shows that TA is the major M. xanthus-diffusible antibacterial agent against E. coli. Correspondingly, the TA(-) mutant was defective in E. coli killing. Separately, an engineered E. coli strain resistant to TA was shown to be resistant toward predation. Exogenous addition of spectinomycin, a bacteriostatic antibiotic, rescued the predation defect of the TA(-) mutant. In contrast, against Micrococcus luteus the TA(-) mutant exhibited no defect in ZOI or killing. Thus, TA plays a selective role on prey species. To extend these studies to other myxobacteria, the role of antibiotic corallopyronin production in predation was tested and also found to be required for Corallococcus coralloides killing on E. coli. Next, a role of TA production in myxobacterial fitness was assessed by measuring swarm expansion. Here, the TA(-) mutant had a specific swarm rate reduction on prey lawns, and thus reduced fitness, compared to an isogenic TA(+) strain. Based on these observations, we conclude that myxobacterial antibiotic production can function as a predatory weapon. To our knowledge, this is the first report to directly show a link between secondary metabolite production and predation.
Larson, R, Messing J.  1982.  Apple II software for M13 shotgun DNA sequencing. Nucleic acids research. 10:39-49. AbstractWebsite
A set of programs is presented for the reconstruction of a DNA sequence from data generated by the M13 shotgun sequencing technique. Once the sequence has been established and stored other programs are used for its analysis. The programs have been written for the Apple II microcomputer. A minimum investment is required for the hardware and the software is easily interchangeable between the growing number of interested researchers. Copies are available in ready to use form.
Dismukes, GC, Carrieri D, Bennette N, Ananyev GM, Posewitz MC.  2008.  Aquatic phototrophs: efficient alternatives to land-based crops for biofuels. Curr Opin Biotechnol. 19:235-40. AbstractWebsite
To mitigate some of the potentially deleterious environmental and agricultural consequences associated with current land-based-biofuel feedstocks, we propose the use of biofuels derived from aquatic microbial oxygenic photoautotrophs (AMOPs), more commonly known as cyanobacteria, algae, and diatoms. Herein we review their demonstrated productivity in mass culturing and aspects of their physiology that are particularly attractive for integration into renewable biofuel applications. Compared with terrestrial crops, AMOPs are inherently more efficient solar collectors, use less or no land, can be converted to liquid fuels using simpler technologies than cellulose, and offer secondary uses that fossil fuels do not provide. AMOPs pose a new set of technological challenges if they are to contribute as biofuel feedstocks.
Parkinson, G, Gunasekera A, Vojtechovsky J, Zhang X, Kunkel TA, Berman H, Ebright RH.  1996.  Aromatic hydrogen bond in sequence-specific protein DNA recognition.. Nature structural biology. 3(10):837-41.
Sheats, JE, Micai K, Bleier S, Storey D, Sellito E, Carrell TG, Maneiro M, Bourles E, Dismukes GC, Rheingold AL et al..  2002.  Assembly of manganese-oxo clusters in solution as models for the photosynthetic oxygen-evolving complex. Macromolecular Symposia. 186:29-34.Website
Pillitteri, LJ, Guo X, Dong J.  2016.  Asymmetric cell division in plants: mechanisms of symmetry breaking and cell fate determination.. Cellular and Molecular Life Sciences. DOI 10.1007/s00018-016-2290-2
Hu, J, Barr MM.  2005.  ATP-2 Interacts with the PLAT Domain of LOV-1 and is Involved in Caenorhabditis Elegans Polycystin Signaling. Mol Biol Cell. 16:458-469. Abstract
Caenorhabditis elegans is a powerful model to study the molecular basis of autosomal dominant polycystic kidney disease (ADPKD). ADPKD is caused by mutations in the polycystic kidney disease (PKD)1 or PKD2 gene, encoding polycystin (PC)-1 or PC-2, respectively. The C. elegans polycystins LOV-1 and PKD-2 are required for male mating behaviors and are localized to sensory cilia. The function of the evolutionarily conserved polycystin/lipoxygenase/alpha-toxin (PLAT) domain found in all PC-1 family members remains an enigma. Here, we report that ATP-2, the beta subunit of the ATP synthase, physically associates with the LOV-1 PLAT domain and that this interaction is evolutionarily conserved. In addition to the expected mitochondria localization, ATP-2 and other ATP synthase components colocalize with LOV-1 and PKD-2 in cilia. Disrupting the function of the ATP synthase or overexpression of atp-2 results in a male mating behavior defect. We further show that atp-2, lov-1, and pkd-2 act in the same molecular pathway. We propose that the ciliary localized ATP synthase may play a previously unsuspected role in polycystin signaling.
Muley, PD, McNeill EM, Marzinke MA, Knobel KM, Barr MM, Clagett-Dame M.  2008.  The atRA-responsive gene Neuron Navigator 2 Functions in Neurite Outgrowth and Axonal Elongation. Dev Neurobiol. 68:1441-1453. Abstract
Neuron navigator 2 (Nav2) was first identified as an all-trans retinoic acid (atRA)-responsive gene in human neuroblastoma cells (retinoic acid-induced in neuroblastoma 1, RAINB1) that extend neurites after exposure to atRA. It is structurally related to the Caenorhabditis elegans unc-53 gene that is required for cell migration and axonal outgrowth. To gain insight into NAV2 function, the full-length human protein was expressed in C. elegans unc-53 mutants under the control of a mechanosensory neuron promoter. Transgene expression of NAV2 rescued the defects in unc-53 mutant mechanosensory neuron elongation, indicating that Nav2 is an ortholog of unc-53. Using a loss-of-function approach, we also show that Nav2 induction is essential for atRA to induce neurite outgrowth in SH-SY5Y cells. The NAV2 protein is located both in the cell body and along the length of the growing neurites of SH-SY5Y cells in a pattern that closely mimics that of neurofilament and microtubule proteins. Transfection of Nav2 deletion constructs in Cos-1 cells reveals a region of the protein (aa 837-1065) that directs localization with the microtubule cytoskeleton. Collectively, this work supports a role for NAV2 in neurite outgrowth and axonal elongation and suggests this protein may act by facilitating interactions between microtubules and other proteins such as neurofilaments that are key players in the formation and stability of growing neurites.
Mathieu, J, Cauvin C, Moch C, Radford SJ, Sampaio P, Perdigoto CN, Schweisguth F, Bardin AJ, Sunkel CE, McKim K et al..  2013.  Aurora B and cyclin B have opposite effects on the timing of cytokinesis abscission in Drosophila germ cells and in vertebrate somatic cells. Dev Cell. 26(3):250-65.Website
Krichevsky, A, Meyers B, Vainstein A, Maliga P, Citovsky V.  2010.  Autoluminescent plants. PloS one. 5:e15461. AbstractWebsite
Prospects of obtaining plants glowing in the dark have captivated the imagination of scientists and layman alike. While light emission has been developed into a useful marker of gene expression, bioluminescence in plants remained dependent on externally supplied substrate. Evolutionary conservation of the prokaryotic gene expression machinery enabled expression of the six genes of the lux operon in chloroplasts yielding plants that are capable of autonomous light emission. This work demonstrates that complex metabolic pathways of prokaryotes can be reconstructed and function in plant chloroplasts and that transplastomic plants can emit light that is visible by naked eye.
Galli, M, Liu Q, Moss BL, Malcomber S, Li W, Gaines C, Federici S, Roshkovan J, Meeley R, Nemhauser J et al..  2015.  Auxin signaling modules regulate maize inflorescence architecture. Proc Natl Acad Sci USA. 112:13372-13377. AbstractWebsite
In plants, small groups of pluripotent stem cells called axillary meristems are required for the formation of the branches and flowers that eventually establish shoot architecture and drive reproductive success. To ensure the proper formation of new axillary meristems, the specification of boundary regions is required for coordinating their development. We have identified two maize genes, BARREN INFLORESCENCE1 and BARREN INFLORESCENCE4 (BIF1 and BIF4), that regulate the early steps required for inflorescence formation. BIF1 and BIF4 encode AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins, which are key components of the auxin hormone signaling pathway that is essential for organogenesis. Here we show that BIF1 and BIF4 are integral to auxin signaling modules that dynamically regulate the expression of BARREN STALK1 (BA1), a basic helix-loop-helix (bHLH) transcriptional regulator necessary for axillary meristem formation that shows a striking boundary expression pattern. These findings suggest that auxin signaling directly controls boundary domains during axillary meristem formation and define a fundamental mechanism that regulates inflorescence architecture in one of the most widely grown crop species.
Chakraborty, A, Wang D, Ebright YW, Ebright RH.  2010.  Azide-specific labeling of biomolecules by Staudinger-Bertozzi ligation phosphine derivatives of fluorescent probes suitable for single-molecule fluorescence spectroscopy.. Methods in enzymology. 472:19-30. Abstract
We describe the synthesis of phosphine derivatives of three fluorescent probes that have a brightness and photostability suitable for single-molecule fluorescence spectroscopy and microscopy: Alexa488, Cy3B, and Alexa647. In addition, we describe procedures for use of these reagents in azide-specific, bioorthogonal labeling through Staudinger-Bertozzi ligation, as well as procedures for the quantitation of labeling specificity and labeling efficiency. The reagents and procedures of this report enable chemoselective, site-selective labeling of azide-containing biomolecules for single-molecule fluorescence spectroscopy and microscopy.
B
Estrem, ST, Ross W, Gaal T, Chen ZW, Niu W, Ebright RH, Gourse RL.  1999.  Bacterial promoter architecture: subsite structure of UP elements and interactions with the carboxy-terminal domain of the RNA polymerase alpha subunit.. Genes & development. 13(16):2134-47. Abstract
We demonstrate here that the previously described bacterial promoter upstream element (UP element) consists of two distinct subsites, each of which, by itself, can bind the RNA polymerase holoenzyme alpha subunit carboxy-terminal domain (RNAP alphaCTD) and stimulate transcription. Using binding-site-selection experiments, we identify the consensus sequence for each subsite. The selected proximal subsites (positions -46 to -38; consensus 5'-AAAAAARNR-3') stimulate transcription up to 170-fold, and the selected distal subsites (positions -57 to -47; consensus 5'-AWWWWWTTTTT-3') stimulate transcription up to 16-fold. RNAP has subunit composition alpha(2)betabeta'sigma and thus contains two copies of alphaCTD. Experiments with RNAP derivatives containing only one copy of alphaCTD indicate, in contrast to a previous report, that the two alphaCTDs function interchangeably with respect to UP element recognition. Furthermore, function of the consensus proximal subsite requires only one copy of alphaCTD, whereas function of the consensus distal subsite requires both copies of alphaCTD. We propose that each subsite constitutes a binding site for a copy of alphaCTD, and that binding of an alphaCTD to the proximal subsite region (through specific interactions with a consensus proximal subsite or through nonspecific interactions with a nonconsensus proximal subsite) is a prerequisite for binding of the other alphaCTD to the distal subsite.
Minakhin, L, Bhagat S, Brunning A, Campbell EA, Darst SA, Ebright RH, Severinov K.  2001.  Bacterial RNA polymerase subunit omega and eukaryotic RNA polymerase subunit RPB6 are sequence, structural, and functional homologs and promote RNA polymerase assembly.. Proceedings of the National Academy of Sciences of the United States of America. 98(3):892-7. Abstract
Bacterial DNA-dependent RNA polymerase (RNAP) has subunit composition beta'betaalpha(I)alpha(II)omega. The role of omega has been unclear. We show that omega is homologous in sequence and structure to RPB6, an essential subunit shared in eukaryotic RNAP I, II, and III. In Escherichia coli, overproduction of omega suppresses the assembly defect caused by substitution of residue 1362 of the largest subunit of RNAP, beta'. In yeast, overproduction of RPB6 suppresses the assembly defect caused by the equivalent substitution in the largest subunit of RNAP II, RPB1. High-resolution structural analysis of the omega-beta' interface in bacterial RNAP, and comparison with the RPB6-RPB1 interface in yeast RNAP II, confirms the structural relationship and suggests a "latching" mechanism for the role of omega and RPB6 in promoting RNAP assembly.
Deighan, P, Diez CM, Leibman M, Hochschild A, Nickels BE.  2008.  The bacteriophage lambda Q antiterminator protein contacts the beta-flap domain of RNA polymerase. Proc Natl Acad Sci U S A. 105:15305-10. AbstractWebsite
The multisubunit RNA polymerase (RNAP) in bacteria consists of a catalytically active core enzyme (alpha(2)beta beta'omega) complexed with a sigma factor that is required for promoter-specific transcription initiation. During early elongation the stability of interactions between sigma and core decreases, in part because of the nascent RNA-mediated destabilization of an interaction between region 4 of sigma and the flap domain of the beta-subunit (beta-flap). The nascent RNA-mediated destabilization of the sigma region 4/beta-flap interaction is required for the bacteriophage lambda Q antiterminator protein (lambdaQ) to engage the RNAP holoenzyme. Here, we provide an explanation for this requirement by showing that lambdaQ establishes direct contact with the beta-flap during the engagement process, thus competing with sigma(70) region 4 for access to the beta-flap. We also show that lambdaQ's affinity for the beta-flap is calibrated to ensure that lambdaQ activity is restricted to the lambda late promoter P(R'). Specifically, we find that strengthening the lambdaQ/beta-flap interaction allows lambdaQ to bypass the requirement for specific cis-acting sequence elements, a lambdaQ-DNA binding site and a RNAP pause-inducing element, that normally ensure lambdaQ is recruited exclusively to transcription complexes associated with P(R'). Our findings demonstrate that the beta-flap can serve as a direct target for regulators of elongation.