scholarly journals Assembly of a tRNA splicing complex: evidence for concerted excision and joining steps in splicing in vitro.

1986 ◽  
Vol 6 (2) ◽  
pp. 635-644 ◽  
Author(s):  
C L Greer

Splicing of tRNA precursors in Saccharomyces cerevisiae extracts proceeds in two steps; excision of the intervening sequence and ligation of the tRNA halves. The ability to resolve these two steps and the distinct physical properties of the endonuclease and ligase suggested that the splicing steps may not be concerted and that these two enzymes may act independently in vivo. A ligase competition assay was developed to examine whether the excision and ligation steps in tRNA splicing in vitro are concerted or independent. The ability of either yeast ligase or T4 ligase plus kinase to join the tRNA halves produced by endonuclease and the distinct structures of the reaction products provided the basis for the competition assay. In control reactions, joining of isolated tRNA halves formed by preincubation with endonuclease was measured. The ratio of yeast to T4 reaction products in these control assays reflected the ratio of the enzyme activities, as would be expected if each has equal access to the substrate. In splicing competition assays, endonuclease and pre-tRNA were added to ligase mixtures, and joining of the halves that were formed was measured. In these assays the products were predominantly those of the yeast ligase even when the T4 enzymes were present in excess. These results demonstrate preferential access of yeast ligase to the endonuclease products and provide evidence for the assembly of a functional tRNA splicing complex in vitro. This observation has important implications for the organization of the splicing components and of the gene expression pathway in vivo.

1986 ◽  
Vol 6 (2) ◽  
pp. 635-644
Author(s):  
C L Greer

Splicing of tRNA precursors in Saccharomyces cerevisiae extracts proceeds in two steps; excision of the intervening sequence and ligation of the tRNA halves. The ability to resolve these two steps and the distinct physical properties of the endonuclease and ligase suggested that the splicing steps may not be concerted and that these two enzymes may act independently in vivo. A ligase competition assay was developed to examine whether the excision and ligation steps in tRNA splicing in vitro are concerted or independent. The ability of either yeast ligase or T4 ligase plus kinase to join the tRNA halves produced by endonuclease and the distinct structures of the reaction products provided the basis for the competition assay. In control reactions, joining of isolated tRNA halves formed by preincubation with endonuclease was measured. The ratio of yeast to T4 reaction products in these control assays reflected the ratio of the enzyme activities, as would be expected if each has equal access to the substrate. In splicing competition assays, endonuclease and pre-tRNA were added to ligase mixtures, and joining of the halves that were formed was measured. In these assays the products were predominantly those of the yeast ligase even when the T4 enzymes were present in excess. These results demonstrate preferential access of yeast ligase to the endonuclease products and provide evidence for the assembly of a functional tRNA splicing complex in vitro. This observation has important implications for the organization of the splicing components and of the gene expression pathway in vivo.


2021 ◽  
Author(s):  
Tague J.G. ◽  
A. Regmi ◽  
G.J. Gregory ◽  
E.F. Boyd

ABSTRACTFis (Factor for Inversion Stimulation) is a global regulator that is highly expressed during exponential growth and undetectable in stationary growth. Quorum sensing (QS) is a global regulatory mechanism that controls gene expression in response to cell density and growth phase. In V. parahaemolyticus, a marine species and a significant human pathogen, the QS regulatory sRNAs, Qrr1 to Qrr5, negatively regulate the high cell density QS master regulator OpaR. OpaR is a positive regulator of capsule polysaccharide (CPS) formation required for biofilm formation and a repressor of swarming motility. In Vibrio parahaemolyticus, we showed, using genetics and DNA binding assays, that Fis bound directly to the regulatory regions of the qrr genes and was a positive regulator of these genes. In the Δfis mutant, opaR expression was induced and a robust CPS and biofilm was produced, while swarming motility was abolished. Expression analysis and promoter binding assays showed that Fis was a direct activator of both the lateral flagellum laf operon and the surface sensing scrABC operon, both required for swarming motility. In in vitro growth competition assays, Δfis was outcompeted by wild type in minimal media supplemented with intestinal mucus, and we showed that Fis directly modulated catabolism gene expression. In in vivo colonization competition assays, Δfis was outcompeted by wild type, indicating Fis is required for fitness. Overall, these data demonstrate a direct role for Fis in QS, motility, and metabolism in V. parahaemolyticus.IMPORTANCEIn this study, we examined the role of Fis in modulating expression of the five-quorum sensing regulatory sRNAs, qrr1 to qrr5, and showed that Fis is a direct positive regulator of QS, which oppositely controls CPS and swarming motility in V. parahaemolyticus. The Δfis deletion mutant was swarming defective due to a requirement for Fis in lateral flagella and surface sensing gene expression. Thus, Fis links QS and surface sensing to control swarming motility and, indirectly, CPS production. Fis was also required for cell metabolism, acting as a direct regulator of several carbon catabolism loci. Both in vitro and in vivo competition assays showed that the Δfis mutant had a significant defect compared to wild type. Overall, our data demonstrates that Fis plays a critical role in V. parahaemolyticus physiology that was previously unexamined.


2008 ◽  
Vol 46 (01) ◽  
Author(s):  
F Moriconi ◽  
H Christiansen ◽  
H Christiansen ◽  
N Sheikh ◽  
J Dudas ◽  
...  

2020 ◽  
Vol 139 ◽  
pp. 153-160
Author(s):  
S Peeralil ◽  
TC Joseph ◽  
V Murugadas ◽  
PG Akhilnath ◽  
VN Sreejith ◽  
...  

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.


2017 ◽  
Vol 95 (3) ◽  
pp. 1313 ◽  
Author(s):  
L. Zhang ◽  
L. F. Schütz ◽  
C. L. Robinson ◽  
M. L. Totty ◽  
L. J. Spicer

2005 ◽  
Vol 83 (4) ◽  
pp. 535-547 ◽  
Author(s):  
Gareth N Corry ◽  
D Alan Underhill

To date, the majority of the research regarding eukaryotic transcription factors has focused on characterizing their function primarily through in vitro methods. These studies have revealed that transcription factors are essentially modular structures, containing separate regions that participate in such activities as DNA binding, protein–protein interaction, and transcriptional activation or repression. To fully comprehend the behavior of a given transcription factor, however, these domains must be analyzed in the context of the entire protein, and in certain cases the context of a multiprotein complex. Furthermore, it must be appreciated that transcription factors function in the nucleus, where they must contend with a variety of factors, including the nuclear architecture, chromatin domains, chromosome territories, and cell-cycle-associated processes. Recent examinations of transcription factors in the nucleus have clarified the behavior of these proteins in vivo and have increased our understanding of how gene expression is regulated in eukaryotes. Here, we review the current knowledge regarding sequence-specific transcription factor compartmentalization within the nucleus and discuss its impact on the regulation of such processes as activation or repression of gene expression and interaction with coregulatory factors.Key words: transcription, subnuclear localization, chromatin, gene expression, nuclear architecture.


2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Ying Xie ◽  
Xiaofeng Hang ◽  
Wensheng Xu ◽  
Jing Gu ◽  
Yuanjing Zhang ◽  
...  

Abstract Background Most of the biological functions of circular RNAs (circRNAs) and the potential underlying mechanisms in hepatocellular carcinoma (HCC) have not yet been discovered. Methods In this study, using circRNA expression data from HCC tumor tissues and adjacent tissues from the Gene Expression Omnibus database, we identified out differentially expressed circRNAs and verified them by qRT-PCT. Functional experiments were performed to evaluate the effects of circFAM13B in HCC in vitro and in vivo. Results We found that circFAM13B was the most significantly differentially expressed circRNA in HCC tissue. Subsequently, in vitro and in vivo studies also demonstrated that circFAM13B promoted the proliferation of HCC. Further studies revealed that circFAM13B, a sponge of miR-212, is involved in the regulation of E2F5 gene expression by competitively binding to miR-212, inhibits the activation of the P53 signalling pathway, and promotes the proliferation of HCC cells. Conclusions Our findings revealed the mechanism underlying the regulatory role played by circFAM13B, miR-212 and E2F5 in HCC. This study provides a new theoretical basis and novel target for the clinical prevention and treatment of HCC.


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