Comparison of promoter-specific events during transcription initiation in mycobacteria

DNA protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter RNAP interactions during transcription initiation in the sigma(A)-dependent promoters P-rrnAPCL1, P-rrnB and P-gyr of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation.

Functional Identification of Regulatory Elements within the Promoter Region of Platelet-Derived Growth Factor 2

Human platelet-derived growth factor (PDGF) is composed of two polypeptide chains, PDGF-1 and PDGF-2,the human homolog of the v-sis oncogene. Deregulation of PDGF-2 expression can confer a growth advantage to cells possessing the cognate receptor and, thus, may contribute to the malignant phenotype. We investigated the regulation of PDGF-2 mRNA expression during megakaryocytic differentiation of K562 cells. Induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) led to a greater than 200-fold increase in PDGF-2 transcript levels in these cells. Induction was dependent on protein synthesis and was not enhanced by cycloheximide exposure.In our initial investigation of the PDGF-2 promoter, a minimal promoter region, which included sequences extending only 42 base pairs upstream of the TATA signal, was found to be as efficient as 4 kilobase pairs upstream of the TATA signal in driving expression of a reporter gene in uninduced K562 cells. We also functionally identified different regulatory sequence elements of the PDGF-2 promoter in TPA-induced K562 cells. One region acted as a transcriptional silencer, while another region was necessary for maximal activity of the promoter in megakaryoblasts. This region was shown to bind nuclear factors and was the target for trans-activation in normal and tumor cells. In one tumor cell line, which expressed high PDGF-2 mRNA levels, the presence of the positive regulatory region resulted in a 30-fold increase in promoter activity. However, the ability of the minimal PDGF-2 promoter to drive reporter gene expression in uninduced K562 cells and normal fibroblasts, which contained no detectable PDGF-2 transcripts, implies the existence of other negative control mechanisms beyond the regulation of promoter activity.

High-quality annotation of promoter regions for 913 bacterial genomes

Motivation: The number of bacterial genomes being sequenced is increasing very rapidly and hence, it is crucial to have procedures for rapid and reliable annotation of their functional elements such as promoter regions, which control the expression of each gene or each transcription unit of the genome. The present work addresses this requirement and presents a generic method applicable across organisms. Results: Relative stability of the DNA double helical sequences has been used to discriminate promoter regions from non-promoter regions. Based on the difference in stability between neighboring regions, an algorithm has been implemented to predict promoter regions on a large scale over 913 microbial genome sequences. The average free energy values for the promoter regions as well as their downstream regions are found to differ, depending on their GC content. Threshold values to identify promoter regions have been derived using sequences flanking a subset of translation start sites from all microbial genomes and then used to predict promoters over the complete genome sequences. An average recall value of 72% (which indicates the percentage of protein and RNA coding genes with predicted promoter regions assigned to them) and precision of 56% is achieved over the 913 microbial genome dataset.

Small Ubiquitin-related Modifier Ligase Activity of Mms21 Is Required for Maintenance of Chromosome Integrity during the Unperturbed Mitotic Cell Division Cycle in Saccharomyces cerevisiae

The SUMO ligase activity of Mms21/Nse2, a conserved member of the Smc5/6 complex, is required for resisting extrinsically induced genotoxic stress. We report that the Mms21 SUMO ligase activity is also required during the unchallenged mitotic cell cycle in Saccharomyces cerevisiae. SUMO ligase-defective cells were slow growing and spontaneously incurred DNA damage. These cells required caffeine-sensitive Mec1 kinase-dependent checkpoint signaling for survival even in the absence of extrinsically induced genotoxic stress. SUMO ligase-defective cells were sensitive to replication stress and displayed synthetic growth defects with DNA damage checkpoint-defective mutants such as mec1, rad9, and rad24. MMS21 SUMO ligase and mediator of replication checkpoint 1 gene (MRC1) were epistatic with respect to hydroxyurea-induced replication stress or methyl methanesulfonate-induced DNA damage sensitivity. Subjecting Mms21 SUMO ligase-deficient cells to transient replication stress resulted in enhancement of cell cycle progression defects such as mitotic delay and accumulation of hyperploid cells. Consistent with the spontaneous activation of the DNA damage checkpoint pathway observed in the Mms21-mediated sumoylation-deficient cells, enhanced frequency of chromosome breakage and loss was detected in these mutant cells. A mutation in the conserved cysteine 221 that is engaged in coordination of the zinc ion in Loop 2 of the Mms21 SPL-RING E3 ligase catalytic domain resulted in strong replication stress sensitivity and also conferred slow growth and Mec1 dependence to unchallenged mitotically dividing cells. Our findings establish Mms21-mediated sumoylation as a determinant of cell cycle progression and maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in budding yeast.

The stretching of single poly-ubiquitin molecules: Static versus dynamic disorder in the non-exponential kinetics of chain unfolding

Static disorder has recently been implicated in the non-exponential kinetics of the unfolding of single molecules of poly-ubiquitin under a constant force Kuo, Garcia-Manyes, Li, Barel, Lu, Berne, Urbakh, Klafter, and Fernandez, Proc. Natl. Acad. Sci. U. S. A. 107, 11336 (2010)]. In the present paper, it is suggested that dynamic disorder may provide a plausible, alternative description of the experimental observations. This suggestion is made on the basis of a model in which the barrier to chain unfolding is assumed to be modulated by a control parameter r that evolves in a parabolic potential under the action of fractional Gaussian noise according to a generalized Langevin equation. The treatment of dynamic disorder within this model is pursued using Zwanzig's indirect approach to noise averaging Acc. Chem. Res. 23, 148 (1990)]. In conjunction with a self-consistent closure scheme developed by Wilemski and Fixman J. Chem. Phys. 58, 4009 (1973); ibid. 60, 866 (1974)], this approach eventually leads to an expression for the chain unfolding probability that can be made to fit the corresponding experimental data very closely. (C) 2011 American Institute of Physics.

Mutations in beta ` subunit of Escherichia coli RNA polymerase perturb the activator polymerase functional interaction required for promoter clearance

P>Transcription activator C employs a unique mechanism to activate mom gene of bacteriophage Mu. The activation process involves, facilitating the recruitment of RNA polymerase (RNAP) by altering the topology of the promoter and enhancing the promoter clearance by reducing the abortive transcription. To understand the basis of this multi-step activation mechanism, we investigated the nature of the physical interaction between C and RNAP during the process. A variety of assays revealed that only DNA-bound C contacts the beta' subunit of RNAP. Consistent to these results, we have also isolated RNAP mutants having mutations in the beta' subunit which were compromised in C-mediated activation. Mutant RNAPs show reduced productive transcription and increased abortive initiation specifically at the C-dependent mom promoter. Positive control (pc) mutants of C, defective in interaction with RNAP, retained the property of recruiting RNAP to the promoter but were unable to enhance promoter clearance. These results strongly suggest that the recruitment of RNAP to the mom promoter does not require physical interaction with C, whereas a contact between the beta' subunit and the activator, and the subsequent allosteric changes in the active site of the enzyme are essential for the enhancement of promoter clearance.

DNA Free Energy-Based Promoter Prediction and Comparative Analysis of Arabidopsis and Rice Genomes

The cis-regulatory regions on DNA serve as binding sites for proteins such as transcription factors and RNA polymerase. The combinatorial interaction of these proteins plays a crucial role in transcription initiation, which is an important point of control in the regulation of gene expression. We present here an analysis of the performance of an in silico method for predicting cis-regulatory regions in the plant genomes of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) on the basis of free energy of DNA melting. For protein-coding genes, we achieve recall and precision of 96% and 42% for Arabidopsis and 97% and 31% for rice, respectively. For noncoding RNA genes, the program gives recall and precision of 94% and 75% for Arabidopsis and 95% and 90% for rice, respectively. Moreover, 96% of the false-positive predictions were located in noncoding regions of primary transcripts, out of which 20% were found in the first intron alone, indicating possible regulatory roles. The predictions for orthologous genes from the two genomes showed a good correlation with respect to prediction scores and promoter organization. Comparison of our results with an existing program for promoter prediction in plant genomes indicates that our method shows improved prediction capability.

Identification of a core promoter and a novel isoform of the human TSCI gene transcript and structural comparison with mouse homolog

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder with loci on chromosome 9q34.12 (TSC1) and chromosome 16p13.3 (TSC2). Genes for both loci have been isolated and characterized. The promoters of both genes have not been characterized so far and little is known about the regulation of these genes. This study reports the characterization of the human TSC1 promoter region for the first time. We have identified a novel alternative isoform in the 5' untranslated region (UTR) of the TSC1 gene transcript involving exon 1. Alternative isoforms in the 5' UTR of the mouse Tsc1 gene transcript involving exon I and exon 2 have also been identified. We have identified three upstream open reading frames (uORFs) in the 5' UTR of the TSC1/Tsc1 gene. A comparative study of the 5' UTR of TSC1/Tsc1 gene has revealed that there is a high degree of similarity not only in the sequence but also in the splicing pattern of both human and mouse TSC1 genes. We have used PCR methodology to isolate approximately 1.6 kb genomic DNA 5' to the TSC1 cDNA. This sequence has directed a high level of expression of luciferase activity in both HeLa and HepG2 cells. Successive 5' and 3' deletion analysis has suggested that a -587 bp region, from position +77 to -510 from the transcription start site (TSS), contains the promoter activity. Interestingly, this region contains no consensus TATA box or CAAT box. However, a 521-bp fragment surrounding the TSS exhibits the characteristics of a CpG island which overlaps with the promoter region. The identification of the TSC1 promoter region will help in designing a suitable strategy to identify mutations in this region in patients who do not show any mutations in the coding regions. It will also help to study the regulation of the TSC1 gene and its role in tumorigenesis. (C) 2003 Elsevier B.V. All rights reserved.

Highly fluorescent GFPm2+-based genome integration-proficient promoter probe vector to study Mycobacterium tuberculosis promoters in infected macrophages

Study of activity of cloned promoters in slow-growing Mycobacterium tuberculosis during long-term growth conditions in vitro or inside macrophages, requires a genome-integration proficient promoter probe vector, which can be stably maintained even without antibiotics, carrying a substrate-independent, easily scorable and highly sensitive reporter gene. In order to meet this requirement, we constructed pAKMN2, which contains mycobacterial codon-optimized gfpm2+ gene, coding for GFPm2+ of highest fluorescence reported till date, mycobacteriophage L5 attP-int sequence for genome integration, and a multiple cloning site. pAKMN2 showed stable integration and expression of GFPm2+ from M. tuberculosis and M. smegmatis genome. Expression of GFPm2+, driven by the cloned minimal promoters of M. tuberculosis cell division gene, ftsZ (MtftsZ), could be detected in the M. tuberculosis/pAKMN2-promoter integrants, growing at exponential phase in defined medium in vitro and inside macrophages. Stable expression from genome-integrated format even without antibiotic, and high sensitivity of detection by flow cytometry and fluorescence imaging, in spite of single copy integration, make pAKMN2 useful for the study of cloned promoters of any mycobacterial species under long-term in vitro growth or stress conditions, or inside macrophages.

Identification and characterization of starvation induced msdgc-1 promoter involved in the c-di-GMP turnover

C-di-GMP Bis-(3'-5')-cyclic-dimeric-guanosine monophosphate], a second messenger is involved in intracellular communication in the bacterial species. As a result several multi-cellular behaviors in both Gram-positive and Gram-negative bacteria are directly linked to the intracellular level of c-di-GMP. The cellular concentration of c-di-GMP is maintained by two opposing activities, diguanylate cyclase (DGC) and phosphodiesterase (PDE-A). In Mycobacterium smegmatis, a single bifunctional protein MSDGC-1 is responsible for the cellular concentration of c-di-GMP. A better understanding of the regulation of c-di-GMP at the genetic level is necessary to control the function of above two activities. In this work, we have characterized the promoter element present in msdgc-1 along with the + 1 transcription start site and identified the sigma factors that regulate the transcription of msdgc-1. Interestingly, msdgc-1 utilizes SigA during the initial phase of growth, whereas near the stationary phase SigB containing RNA polymerase takes over the expression of msdgc-1. We report here that the promoter activity of msdgc-1 increases during starvation or depletion of carbon source like glucose or glycerol. When msdgc-1 is deleted, the numbers of viable cells are similar to 10 times higher in the stationary phase in comparison to that of the wild type. We propose here that msdgc-1 is involved in the regulation of cell population density. (C) 2013 Elsevier B.V. All rights reserved.

DNA STRUCTURAL FEATURES AND ARCHITECTURE OF PROMOTER REGIONS PLAY A ROLE IN GENE RESPONSIVENESS OF S. CEREVISIAE

Gene expression is the most fundamental biological process, which is essential for phenotypic variation. It is regulated by various external (environment and evolution) and internal (genetic) factors. The level of gene expression depends on promoter architecture, along with other external factors. Presence of sequence motifs, such as transcription factor binding sites (TFBSs) and TATA-box, or DNA methylation in vertebrates has been implicated in the regulation of expression of some genes in eukaryotes, but a large number of genes lack these sequences. On the other hand, several experimental and computational studies have shown that promoter sequences possess some special structural properties, such as low stability, less bendability, low nucleosome occupancy, and more curvature, which are prevalent across all organisms. These structural features may play role in transcription initiation and regulation of gene expression. We have studied the relationship between the structural features of promoter DNA, promoter directionality and gene expression variability in S. cerevisiae. This relationship has been analyzed for seven different measures of gene expression variability, along with two different regulatory effect measures. We find that a few of the variability measures of gene expression are linked to DNA structural properties, nucleosome occupancy, TATA-box presence, and bidirectionality of promoter regions. Interestingly, gene responsiveness is most intimately correlated with DNA structural features and promoter architecture.

Two-step Synthesis of MoS2 Nanotubes using Shock Waves with Lead as Growth Promoter

A new two-step procedure for the synthesis of MoS2 nanotubes using lead as a growth promoter is reported. In the first step, molybdenum suboxide nanowhiskers containing a small amount of lead atoms were created by exposing a pressed MoS2+Pb mixture to highly compressed shock-heated argon gas, with estimated temperatures exceeding 9900 K. In the second step, these molybdenum suboxide nanowhiskers served as templates for the sulfidization of the oxide into MoS2 nanotubes (by using H2S gas in a reducing atmosphere at 820 degrees C). Unlike the case of WS2 nanotubes, the synthesis of a pure phase of MoS2 nanotubes from molybdenum oxide has proven challenging, due mostly to the volatile nature of the latter at the high requisite reaction temperatures (>800 degrees C). In contrast, the nature and apparent reaction mechanism of the method reported herein are amenable to future scale-up. The high-temperature shockwave system should also facilitate the synthesis of new nanostructures from other layered materials.

Evidence that TSC2 acts as a transcription factor and binds to and represses the promoter of Epiregulin

The TSC2 gene, mutated in patients with tuberous sclerosis complex (TSC), encodes a 200 kDa protein TSC2 (tuberin). The importance of TSC2 in the regulation of cell growth and proliferation is irrefutable. TSC2 in complex with TSC1 negatively regulates the mTOR complex 1 (mTORC1) via RHEB in the PI3K-AKT-mTOR pathway and in turn regulates cell proliferation. It shows nuclear as well as cytoplasmic localization. However, its nuclear function remains elusive. In order to identify the nuclear function of TSC2, a whole-genome expression profiling of TSC2 overexpressing cells was performed, and the results showed differential regulation of 266 genes. Interestingly, transcription was found to be the most populated functional category. EREG (Epiregulin), a member of the epidermal growth factor family, was found to be the most downregulated gene in the microarray analysis. Previous reports have documented elevated levels of EREG in TSC lesions, making its regulatory aspects intriguing. Using the luciferase reporter, ChIP and EMSA techniques, we show that TSC2 binds to the EREG promoter between -352 bp and -303 bp and negatively regulates its expression. This is the first evidence for the role of TSC2 as a transcription factor and of TSC2 binding to the promoter of any gene.

Dissecting Ubiquitin Folding Using the Self-Organized Polymer Model

Folding of Ubiquitin (Ub), a functionally important protein found in eukaryotic organisms, is investigated at low and neutral pH at different temperatures using simulations of the coarse-grained self-organized-polymer model with side chains (SOP-SC). The melting temperatures (T-m's), identified with the peaks in the heat capacity curves, decrease as pH decreases, in qualitative agreement with experiments. The calculated radius of gyration, showing dramatic variations with pH, is in excellent agreement with scattering experiments. At T-m Ub folds in a two-state manner at low and neutral pH. Clustering analysis of the conformations sampled in equilibrium folding trajectories at T-m with multiple transitions between the folded and unfolded states, shows a network of metastable states connecting the native and unfolded states. At low and neutral pH, Ub folds with high probability through a preferred set of conformations resulting in a pH-dependent dominant folding pathway. Folding kinetics reveal that Ub assembly at low pH occurs by multiple pathways involving a combination of nucleation-collapse and diffusion collision mechanism. The mechanism by which Ub folds is dictated by the stability of the key secondary structural elements responsible for establishing long-range contacts and collapse of Ub. Nucleation collapse mechanism holds if the stability of these elements are marginal, as would be the case at elevated temperatures. If the lifetimes associated with these structured microdomains are on the order of hundreds of microseconds, then Ub folding follows the diffusion collision mechanism with intermediates, many of which coincide with those found in equilibrium. Folding at neutral pH is a sequential process with a populated intermediate resembling that sampled at equilibrium. The transition state structures, obtained using a P-fold analysis, are homogeneous and globular with most of the secondary and tertiary structures being native-like. Many of our findings for both the thermodynamics and kinetics of folding are not only in agreement with experiments but also provide missing details not resolvable in standard experiments. The key prediction that folding mechanism varies dramatically with pH is amenable to experimental tests.