Co-evolution of specific amino acid in sigma 1.2 region and nucleotide base in the discriminator to act as sensors of small molecule effectors of transcription initiation in mycobacteria

The transcription from rrn and a number of other promoters is regulated by initiating ribonucleotides (iNTPs) and guanosine tetra/penta phosphate (p)ppGpp], either by strengthening or by weakening of the RNA polymerase (RNAP)-promoter interactions during initiation. Studies in Escherichia coli revealed the importance of a sequence termed discriminator, located between -10 and the transcription start site of the responsive promoters in this mode of regulation. Instability of the open complex at these promoters is attributed to the lack of stabilizing interactions between the suboptimal discriminator and the 1.2 region of sigma 70 (Sig70) in RNAP holoenzyme. We demonstrate a different pattern of interaction between the promoters and sigma A (SigA) of Mycobacterium tuberculosis to execute similar regulation. Instead of cytosine and methionine, thymine at three nucleotides downstream to -10 element and leucine 232 in SigA are found to be essential for iNTPs and pppGpp mediated response at the rrn and gyr promoters of the organism. The specificity of the interaction is substantiated by mutational replacements, either in the discriminator or in SigA, which abolish the nucleotide mediated regulation in vitro or in vivo. Specific yet distinct bases and the amino acids appear to have co-evolved' to retain the discriminator-sigma 1.2 region regulatory switch operated by iNTPs/pppGpp during the transcription initiation in different bacteria.

Role of DNA sequence based structural features of promoters in transcription initiation and gene expression

D Regulatory information for transcription initiation is present in a stretch of genomic DNA, called the promoter region that is located upstream of the transcription start site (TSS) of the gene. The promoter region interacts with different transcription factors and RNA polymerase to initiate transcription and contains short stretches of transcription factor binding sites (TFBSs), as well as structurally unique elements. Recent experimental and computational analyses of promoter sequences show that they often have non-B-DNA structural motifs, as well as some conserved structural properties, such as stability, bendability, nucleosome positioning preference and curvature, across a class of organisms. Here, we briefly describe these structural features, the differences observed in various organisms and their possible role in regulation of gene expression.

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.

Accurate transcription initiation by RNA polymerase II from Candida utilis

An in vitro transcription system from Candida utilis is described. The template used is a hybrid plasmid containing Saccharomyces cerevisiae CYC1 promoter linked to a synthetic 377-bp G-minus casette (1). In vitro transcriptions are carried out in the presence of RNase. T1. Under these conditions only the transcripts that are resistant to RNase T1 accumulate. Using this protocol, it has been shown that in the absence of cytosolic factors RNA polymerase II (pol II) from C. utilis initiated RNA synthesis randomly. But both C. utilis and S. cerevisiae cell-free extracts could direct pol II from C. utilis to initiate transcription accurately. Results also indicated that the general transcription factors are functionally interchangeable between S. cerevisiae and C. utilis

Distinct and Contrasting Transcription Initiation Patterns at Mycobacterium tuberculosis Promoters

Although sequencing of Mycobacterium tuberculosis genome lead to better understanding of transcription units and gene functions, interactions occurring during transcription initiation between RNA polymerase and promoters is yet to be elucidated. Different stages of transcription initiation include promoter specific binding of RNAP, isomerization, abortive initiation and promoter clearance. We have now analyzed these events with four promoters of M. tuberculosis viz. P-gyrB1, P-gyrR, P-rrnPCL1 and P-metU. The promoters differed from each other in their rates of open complex formation, decay, promoter clearance and abortive transcription. The equilibrium binding and kinetic studies of various steps revealed distinct rate limiting events for each of the promoter, which also differed markedly in their characteristics from the respective promoters of Mycobacterium smegmatis. Surprisingly, the transcription at gyr promoter was enhanced in the presence of initiating nucleotides and decreased in the presence of alarmone, pppGpp, a pattern typically seen with rRNA promoters studied so far. The gyr promoter of M. smegmatis, on the other hand, was not subjected to pppGpp mediated regulation. The marked differences in the transcription initiation pathway seen with rrn and gyr promoters of M. smegmatis and M. tuberculosis suggest that such species specific differences in the regulation of expression of the crucial housekeeping genes could be one of the key determinants contributing to the differences in growth rate and lifestyle of the two organisms. Moreover, the distinct rate limiting steps during transcription initiation of each one of the promoters studied point at variations in their intracellular regulation.

Regulation of transcription initiation in mycobacteria

The success of Mycobacterium tuberculosis as a deadly pathogen lies in its ability to survive under adverse conditions during pre- and post-infectious stages. The transcription process and the regulation of gene expression are central to the survival of the pathogen through the harsh conditions. Multiple sigma factors, transcription regulators, diverse two-component systems contribute in tailoring the events to meet the challenges faced by the pathogen. Although the machinery is conserved, many aspects of transcription and its regulation seem to be different in mycobacteria when compared to the other well-studied organisms. Here, we discuss salient aspects of transcription and its regulation in the context of distinct physiology of mycobacteria.

The potential role of a late gene expression factor, lef2, from Bombyx mori nuclear polyhedrosis virus in very late gene transcription and DNA replication

Several late gene expression factors (Lefs) have been implicated in fostering high levels of transcription from the very late gene promoters of polyhedrin and p10 from baculoviruses. We cloned and characterized from Bombyx mori nuclear polyhedrosis virus a late gene expression factor (Bmlef2) that encodes a 209-amino-acid protein harboring a Cys-rich C-terminal domain. The temporal transcription profiles of lef2 revealed a 1.2-kb transcript in both delayed early and late periods after virus infection. Transcription start site mapping identified the presence of an aphidicolin-sensitive late transcript arising from a TAAG motif located at -352 nucleotides and an aphidicolin-insensitive early transcript originating from a TTGT motif located 35 nucleotides downstream to a TATA box at -312 nucleotides, with respect to the +1 ATG of lef2. BmLef2 trans-activated very late gene expression from both polyhedrin and p10 promoters in transient expression assays. Internal deletion of the Cys-rich domain from the C-terminal region abolished the transcriptional activation. Inactivation of Lef2 synthesis by antisense lef2 transcripts drastically reduced the very late gene transcription but showed little effect on the expression from immediate early promoter. Decrease in viral DNA synthesis and a reduction in virus titer were observed only when antisense lef2 was expressed under the immediate early (ie-1) promoter. Furthermore, the antisense experiments suggested that lef2 plays a direct role in very late gene transcription.

Chemistry and biology of DNA-binding small molecules

Regulation of the transcription machinery is one of the many ways to achieve control of gene expression. This has been done either at the transcription initiation stage or at the elongation stage. Different methodologies are known to inhibit transcription initiation via targeting of double-stranded (ds) DNA by: (i) synthetic oligonucleotides, (ii) ds-DNA-specific, sequenceselective minor-groove binders (distamycin A), intercalators (daunomycin) combilexins and (iii) small molecule (peptide or intercalator)-oligonucleotide conjugates. In some cases, instead of ds-DNA, higher order G-quadruplex structures are formed at the start site of transcription. In this regard G-quadruplex DNA-specific small molecules play a significant role towards inhibition of the transcription machinery. Different types of designer DNA-binding agents act as powerful sequence-specific gene modulators, by exerting their effect from transcription regulation to gene modification. But most of these chemotherapeutic agents have serious side effects. Accordingly, there is always a challenge to design such DNA-binding molecules that should not only achieve maximum specific DNA-binding affinity, and cellular and nuclear transport activity, but also would not interfere with the functions of normal cells.

Involvement of S-cerevisiae Rpb4 in subset of pathways related to transcription elongation

Yeast Rpb4, a subunit of RNA pol II is not essential for viability but is involved in multiple cellular phenotypes such as temperature sensitivity, enhanced pseudohyphal morphology, and decreased sporulation. Both in vivo and in vitro studies strongly support involvement of Rpb4 in transcription initiation, while its role in transcription elongation is not entirely consistent. Here we show that Rpb4 is not required for recruitment of RNA pol II on the coding region of YLR454w, a representative long gene. Yet we find strong genetic interaction of rpb4 Delta with mutants in many transcription elongation factors such as Paf1, Spt4, Dst1, Elp3 and Rpb9. We demonstrate that, Rpb4 interacts functionally with Paf1 to affect the transcription elongation of the FKS1 gene. Our results suggest that while Rpb4 is not required for general transcription elongation, it could support transcription elongation for specific of class of genes by interaction with other elongation factors. (C) 2014 Elsevier B.V. All rights reserved.

Relative stability of DNA as a generic criterion for promoter prediction: whole genome annotation of microbial genomes with varying nucleotide base composition

The rapid increase in genome sequence information has necessitated the annotation of their functional elements, particularly those occurring in the non-coding regions, in the genomic context. Promoter region is the key regulatory region, which enables the gene to be transcribed or repressed, but it is difficult to determine experimentally. Hence an in silico identification of promoters is crucial in order to guide experimental work and to pin point the key region that controls the transcription initiation of a gene. In this analysis, we demonstrate that while the promoter regions are in general less stable than the flanking regions, their average free energy varies depending on the GC composition of the flanking genomic sequence. We have therefore obtained a set of free energy threshold values, for genomic DNA with varying GC content and used them as generic criteria for predicting promoter regions in several microbial genomes, using an in-house developed tool `PromPredict'. On applying it to predict promoter regions corresponding to the 1144 and 612 experimentally validated TSSs in E. coli (50.8% GC) and B. subtilis (43.5% GC) sensitivity of 99% and 95% and precision values of 58% and 60%, respectively, were achieved. For the limited data set of 81 TSSs available for M. tuberculosis (65.6% GC) a sensitivity of 100% and precision of 49% was obtained.

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.

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.

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the omega Subunit

The widely conserved omega subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of omega is known to be substituted by GroEL in omega-null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of omega variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to omega, which is largely unstructured, omega mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, omega(6) bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that omega(6) binds to beta ` subunit in vitro with greater affinity than that of omega. The reconstituted RNAP holoenzyme in the presence of omega(6) in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant omega results in death of the cell. Furthermore, lethality of omega(6) is relieved in cells expressing the rpoC2112 allele encoding beta ` (2112), a variant beta ` bearing Y457S substitution, immediately adjacent to the beta ` catalytic center. Our results suggest that the enhanced omega(6)-beta ` interaction may perturb the plasticity of the RNAP active center, implicating a role for omega and its flexible state.