scholarly journals Effects of osmolytes and macromolecular crowders on stable GAAA tetraloops and their preference for a CG closing base pair

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4236
Author(s):  
Kaethe N. Leonard ◽  
Joshua M. Blose
Keyword(s):  
Secondary Structure ◽  
Base Pair ◽  
Structural Changes ◽  
Tertiary Structure ◽  
Vis Spectroscopy ◽  
Rna Tertiary Structure ◽  
The Stability ◽  
And Function ◽  
Peg 8000

Osmolytes and macromolecular crowders have the potential to influence the stability of secondary structure motifs and alter preferences for conserved nucleic acid sequences in vivo. To further understand the cellular function of RNA we observed the effects of a model osmolyte, polyethylene glycol (PEG) 200, and a model macromolecular crowding agent, PEG 8000, on the GAAA tetraloop motif. GAAA tetraloops are conserved, stable tetraloops, and are critical participants in RNA tertiary structure. They also have a thermodynamic preference for a CG closing base pair. The thermal denaturation of model hairpins containing GAAA loops was monitored using UV-Vis spectroscopy in the presence and absence of PEG 200 or PEG 8000. Both of the cosolutes tested influenced the thermodynamic preference for a CG base pair by destabilizing the loop with a CG closing base pair relative to the loop with a GC closing base pair. This result also extended to a related DNA triloop, which provides further evidence that the interactions between the loop and closing base pair are identical for the d(GCA) triloop and the GAAA tetraloop. Our results suggest that in the presence of model PEG molecules, loops with a GC closing base pair may retain some preferential interactions with the cosolutes that are lost in the presence of the CG closing base pair. These results reveal that relatively small structural changes could influence how neutral cosolutes tune the stability and function of secondary structure motifs in vivo.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits

1993 ◽  
Vol 13 (5) ◽  
pp. 2835-2845
Author(s):  
M Deshmukh ◽  
Y F Tsay ◽  
A G Paulovich ◽  
J L Woolford
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Subunit ◽  
Single Copy ◽  
5S Rrna ◽  
Gene Encoding ◽  
Ribosomal Subunits ◽  
Ribosomal Protein L1 ◽  
The Stability ◽  
And Function

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

scholarly journals The 5′ NAD Cap of RNAIII Modulates Toxin Production in Staphylococcus aureus Isolates

2019 ◽  
Vol 202 (6) ◽  
Author(s):  
Hector Gabriel Morales-Filloy ◽  
Yaqing Zhang ◽  
Gabriele Nübel ◽  
Shilpa Elizabeth George ◽  
Natalya Korn ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Secondary Structure ◽  
Quorum Sensing ◽  
Opportunistic Pathogen ◽  
Toxin Production ◽  
Dual Function ◽  
Content Type ◽  
The Stability

ABSTRACT Nicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5′ ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here, we report the occurrence of several NAD-RNAs in the opportunistic pathogen Staphylococcus aureus. Most prominently, RNAIII, a central quorum-sensing regulator of this bacterium’s physiology, was found to be 5′ NAD capped in a range from 10 to 35%. NAD incorporation efficiency into RNAIII was found to depend in vivo on the −1 position of the P3 promoter. An increase in RNAIII’s NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects seem to be caused neither by changes in RNAIII’s secondary structure nor by a different translatability upon NAD attachment, as indicated by unaltered patterns in in vitro chemical probing and toeprinting experiments. Even though we did not observe any effect of this modification on RNAIII’s secondary structure or translatability in vitro, additional unidentified factors might account for the modulation of exotoxins in vivo. Ultimately, the study constitutes a step forward in the discovery of new roles of the NAD molecule in bacteria. IMPORTANCE Numerous organisms, including bacteria, are endowed with a 5′ NAD cap in specific RNAs. While the presence of the 5′ NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5′ NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating the bacterium’s virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.

Use of mutant RNAs in studies on yeast 5S rRNA structure and function

1991 ◽  
Vol 69 (4) ◽  
pp. 217-222 ◽  
Author(s):  
Ross N. Nazar ◽  
Don I. Van Ryk ◽  
Yoon Lee ◽  
C. David Guyer
Keyword(s):  
Structural Changes ◽  
Growth Conditions ◽  
5S Rrna ◽  
Rrna Genes ◽  
Shuttle Vectors ◽  
5S Rrna Genes ◽  
And Function ◽  
Optimized Conditions ◽  
Rna Concentration

The expression of mutant yeast 5S rRNA genes in vivo is reviewed as a basis for further studies on the structure, function, and regulation of the ribosomal 5S rRNA. Specific base substitutions, insertions, or deletions can result in substantial structural changes which can be detected readily by gel electrophoresis, permitting the assay of mutant RNA synthesis and utilization. Furthermore, the use of high and low copy shuttle vectors, as well as alternate growth conditions, permits a wide adjustment of the mutant RNA concentration. Under optimized conditions more than 80% of the cell's RNA can be replaced with mutant molecules. The application of this strategy to studies on the biosynthesis and structure of the 5S rRNA are demonstrated through recently isolated mutations.Key words: site-specific mitogenesis, 5S RNA, ribosomes, yeast transformation.

scholarly journals Genetically tunable frustration controls allostery in an intrinsically disordered transcription factor

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jing Li ◽  
Jordan T White ◽  
Harry Saavedra ◽  
James O Wrabl ◽  
Hesam N Motlagh ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Structural Changes ◽  
Intrinsically Disordered Proteins ◽  
Tertiary Structure ◽  
Control Function ◽  
Disordered Proteins ◽  
Intrinsically Disordered ◽  
Novel Strategy ◽  
Structured Proteins

Intrinsically disordered proteins (IDPs) present a functional paradox because they lack stable tertiary structure, but nonetheless play a central role in signaling, utilizing a process known as allostery. Historically, allostery in structured proteins has been interpreted in terms of propagated structural changes that are induced by effector binding. Thus, it is not clear how IDPs, lacking such well-defined structures, can allosterically affect function. Here, we show a mechanism by which an IDP can allosterically control function by simultaneously tuning transcriptional activation and repression, using a novel strategy that relies on the principle of ‘energetic frustration’. We demonstrate that human glucocorticoid receptor tunes this signaling in vivo by producing translational isoforms differing only in the length of the disordered region, which modulates the degree of frustration. We expect this frustration-based model of allostery will prove to be generally important in explaining signaling in other IDPs.

Contrasting effects of simulated microgravity with and without daily −Gx gravitation on structure and function of cerebral and mesenteric small arteries in rats

2009 ◽  
Vol 107 (6) ◽  
pp. 1710-1721 ◽  
Author(s):  
Le-Jian Lin ◽  
Fang Gao ◽  
Yun-Gang Bai ◽  
Jun-Xiang Bao ◽  
Xiao-Feng Huang ◽  
...  
Keyword(s):  
Structural Changes ◽  
Cerebral Arteries ◽  
Myogenic Tone ◽  
Resistance Arteries ◽  
Cross Sectional ◽  
Small Arteries ◽  
Functional Studies ◽  
Cell Layers ◽  
And Function

This study was designed to test the hypothesis that a 28-day tail suspension (SUS) could induce hypertrophy and enhanced myogenic and vasoconstrictor reactivity in middle cerebral arteries (MCAs), whereas atrophy and decreased myogenic and vasoconstrictor responses in mesenteric third-order arterioles (MSAs). Also, in addition to the functional enhancement in MCAs, structural changes in both kinds of arteries and functional decrement in MSAs could all be prevented by the intervention of daily 1-h dorsoventral (−Gx) gravitation by restoring to standing posture. To test this hypothesis, vessel diameters to pressure alterations and nonreceptor- and receptor-mediated agonists were determined using a pressure arteriograph with a procedure to measure in vivo length and decrease hysteresis of vessel segments and longitudinal middlemost sections of vessels fixed at maximally dilated state were examined using electron microscopy and histomorphometry. Functional studies showed that 28-day tail-suspended, head-down tilt (SUS) resulted in enhanced and decreased myogenic tone and vasoconstrictor responses, respectively, in MCAs and MSAs. Histomorphometric data revealed that SUS-induced hypertrophic changes in MCAs characterized by increases in thickness (T) and cross-sectional area (CSA) of the media and the number of vascular smooth-muscle-cell layers (NCL), whereas in MSAs, it induced decreases in medial CSA and T and NCL. Daily 1-h −Gx over 28 days can fully prevent these differential structural changes in both kinds of small arteries and the functional decrement in MSAs, but not the augmented myogenic tone and increased vasoreactivity in the MCAs. These findings have revealed special features of small resistance arteries during adaptation to microgravity with and without gravity-based countermeasure.

scholarly journals Quality Assurance of Commercial Insulin Formulations: Novel Assay Using Infrared Spectroscopy

2020 ◽  
pp. 193229682091387 ◽  
Author(s):  
Sven Delbeck ◽  
H. Michael Heise
Keyword(s):  
Secondary Structure ◽  
Structural Changes ◽  
Elevated Temperatures ◽  
Tertiary Structure ◽  
Total Reflection ◽  
Attenuated Total Reflection ◽  
Insulin Degradation ◽  
Reflection Spectroscopy ◽  
Animal Testing ◽  
Absorption Bands

Background: For insulins in commercial formulations, degradation can be observed within the certified shelf life when not stored at recommended conditions. Elevated temperatures and exposure to shear forces can cause changes in the secondary structure of the hormone, leading to a decrease in pharmaceutical potency. International pharmacopoeia recommendations for insulin quality monitoring assays mainly rely on liquid chromatography methods. These methods are unable to distinguish between active and inactive forms, both of which may exist in pharmaceutical insulins exposed to stress conditions. Method: Infrared attenuated total reflection spectroscopy has been used for the analysis of insulin dry film preparations using affordable instrumentation. This method can be applied to either formulated insulin specimens or pure insulins obtained by ultrafiltration. Such samples have been stored under different temperatures (0°C, 20°C, and 37°C), and degradation processes have been monitored up to a period of a few months. Results: By analyzing specific shifts of absorption bands in the infrared spectra, which are sensitive to the protein secondary structure, even small structural changes in the hormone become evident. Another option is amide I band deconvolution into individual bands, which can be attributed to secondary structure subunits that are part of the insulin tertiary structure. Conclusion: A novel and innovative method based on infrared attenuated total reflection spectroscopy of insulin dry films is a promising analytical tool for quantifying the degree of insulin degradation, as it provides information on indicating a decrease in biological potency. The established methods for insulin potency assays require animal testing or clamp experiments on people with diabetes.

scholarly journals Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits.

1993 ◽  
Vol 13 (5) ◽  
pp. 2835-2845 ◽  
Author(s):  
M Deshmukh ◽  
Y F Tsay ◽  
A G Paulovich ◽  
J L Woolford
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Subunit ◽  
Single Copy ◽  
5S Rrna ◽  
Gene Encoding ◽  
Ribosomal Subunits ◽  
Ribosomal Protein L1 ◽  
The Stability ◽  
And Function

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

scholarly journals Piv Site-Specific Invertase Requires a DEDD Motif Analogous to the Catalytic Center of the RuvC Holliday Junction Resolvases

2005 ◽  
Vol 187 (10) ◽  
pp. 3431-3437 ◽  
Author(s):  
John M. Buchner ◽  
Anne E. Robertson ◽  
David J. Poynter ◽  
Shelby S. Denniston ◽  
Anna C. Karls
Keyword(s):  
Catalytic Activity ◽  
Tertiary Structure ◽  
Rnase H ◽  
Holliday Junction ◽  
Site Specific ◽  
Acidic Residues ◽  
Insertion Elements ◽  
The Stability ◽  
Retroviral Integrase

ABSTRACT Piv, a unique prokaryotic site-specific DNA invertase, is related to transposases of the insertion elements from the IS110/IS492 family and shows no similarity to the site-specific recombinases of the tyrosine- or serine-recombinase families. Piv tertiary structure is predicted to include the RNase H-like fold that typically encompasses the catalytic site of the recombinases or nucleases of the retroviral integrase superfamily, including transposases and RuvC-like Holliday junction resolvases. Analogous to the DDE and DEDD catalytic motifs of transposases and RuvC, respectively, four Piv acidic residues D9, E59, D101, and D104 appear to be positioned appropriately within the RNase H fold to coordinate two divalent metal cations. This suggests mechanistic similarity between site-specific inversion mediated by Piv and transposition or endonucleolytic reactions catalyzed by enzymes of the retroviral integrase superfamily. The role of the DEDD motif in Piv catalytic activity was addressed using Piv variants that are substituted individually or multiply at these acidic residues and assaying for in vivo inversion, intermolecular recombination, and DNA binding activities. The results indicate that all four residues of the DEDD motif are required for Piv catalytic activity. The DEDD residues are not essential for inv recombination site recognition and binding, but this acidic tetrad does appear to contribute to the stability of Piv-inv interactions. On the basis of these results, a working model for Piv-mediated inversion that includes resolution of a Holliday junction is presented.

Effect of Zn(II) on the Structure and Biological Activity of Natural β-NGF

2004 ◽  
Vol 36 (2) ◽  
pp. 99-104 ◽  
Author(s):  
Guang-Hua Zhao ◽  
Ping Yu ◽  
Xiao-Song Hu ◽  
Lei Zhao
Keyword(s):  
Biological Activity ◽  
Secondary Structure ◽  
Atomic Absorption ◽  
Absorption Spectroscopy ◽  
Atomic Absorption Spectroscopy ◽  
Structural Changes ◽  
Tertiary Structure ◽  
Zinc Ion ◽  
Stopped Flow ◽  
Flameless Atomic Absorption Spectroscopy

Abstract Only β-NGF, the subunit of the 7S NGF complex, exhibits NGF activity, but the function of the zinc ion in native β-NGF has received little attention. Flameless atomic absorption spectroscopy (FAAS) measurements reveal that native β-NGF contains Zn(II) with a Zn(II)/β-NGF stoichiometry of 1:14.6. The presence of Zn(II) in the native molecule results in significant changes of the secondary structure and local tertiary structure around Trp(s) with respect to those of apo β-NGF, as suggested by spectra of fluorescence and circular dichrosim. Stopped-flow studies show that there are at least two steps during the interaction of Zn(II) with the apo form. In comparison with its apo form, the native β-NGF shows a higher ability to trigger the proliferation of TF1 cells and mediate the survival of PC12. Thus it is most likely that the structural changes caused by the presence of Zn(II) directly lead to the increase in the biological activity of β-NGF. All results indicate that Zn(II) in native β-NGF plays an important role in the structure and the biological activity of the protein.

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