scholarly journals Impacts of Fluorescent Base Analogue Substitution on the Folding of a Riboswitch

2021 ◽  
Author(s):  
Janson E Hoeher ◽  
Michael A Veirs ◽  
Julia R Widom
Keyword(s):  
Critical Role ◽  
Binding Pocket ◽  
Ligand Recognition ◽  
Base Pairs ◽  
Stem Loop ◽  
Pseudoknot Structure ◽  
Expression Platform ◽  
Stem Loop Structure ◽  
Aptamer Domain

Riboswitches are gene-regulating mRNA segments most commonly found in bacteria. A riboswitch contains an aptamer domain that binds to a ligand, causing a conformational change in a downstream expression platform. The aptamer domain of the Class I preQ1 riboswitch from Bacillus subtilis, which consists of a stem-loop structure and an adenine-rich single-stranded tail (L3), re-folds into a pseudoknot structure upon binding of its ligand, preQ1. To study the role of L3 in ligand recognition, we inserted 2-aminopurine (2-AP), a fluorescent base analogue of adenine (A), into the riboswitch at six different positions within L3. 2-AP differs from A in the relocation of its amino group from C6 to C2, allowing us to directly probe the significance of this specific functional group. We used circular dichroism spectroscopy and thermal denaturation experiments to study the structure and stability, respectively, of the riboswitch in the absence and presence of preQ1. At all labeling positions tested, 2-AP substitution inhibited the ability of preQ1 to stabilize the pseudoknot structure, with its location impacting the severity of the effect. Structural studies of the riboswitch suggest that at the most detrimental labeling sites, 2-AP substitution disrupts non-canonical base pairs. Our results show that these base pairs and tertiary interactions involving other residues in L3 play a critical role in ligand recognition by the preQ1 riboswitch, even at positions that are distal to the ligand binding pocket. They also highlight the importance of accounting for perturbations that fluorescent analogues like 2-AP may exert on the system being studied.

scholarly journals Ribosomal pausing during translation of an RNA pseudoknot.

1993 ◽  
Vol 13 (11) ◽  
pp. 6931-6940 ◽  
Author(s):  
P Somogyi ◽  
A J Jenner ◽  
I Brierley ◽  
S C Inglis
Keyword(s):  
High Efficiency ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Stem Loop ◽  
Pseudoknot Structure ◽  
Stem Loop Structure ◽  
Dead End ◽  
Rna Pseudoknot ◽  
Slippery Sequence

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

scholarly journals Ribosomal pausing during translation of an RNA pseudoknot

1993 ◽  
Vol 13 (11) ◽  
pp. 6931-6940 ◽  
Author(s):  
P Somogyi ◽  
A J Jenner ◽  
I Brierley ◽  
S C Inglis
Keyword(s):  
High Efficiency ◽  
Mutational Analysis ◽  
Base Pairs ◽  
Stem Loop ◽  
Pseudoknot Structure ◽  
Stem Loop Structure ◽  
Dead End ◽  
Rna Pseudoknot ◽  
Slippery Sequence

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

scholarly journals Terminal uridyltransferase 7 regulates TLR4-triggered inflammation by controlling Regnase-1 mRNA uridylation and degradation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chia-Ching Lin ◽  
Yi-Ru Shen ◽  
Chi-Chih Chang ◽  
Xiang-Yi Guo ◽  
Yun-Yun Young ◽  
...  
Keyword(s):  
Posttranscriptional Regulation ◽  
Inflammatory Responses ◽  
Rna Stability ◽  
Innate Immune ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
Tlr4 Activation ◽  
Harmful Effects ◽  
Different Levels

AbstractDifferent levels of regulatory mechanisms, including posttranscriptional regulation, are needed to elaborately regulate inflammatory responses to prevent harmful effects. Terminal uridyltransferase 7 (TUT7) controls RNA stability by adding uridines to its 3′ ends, but its function in innate immune response remains obscure. Here we reveal that TLR4 activation induces TUT7, which in turn selectively regulates the production of a subset of cytokines, including Interleukin 6 (IL-6). TUT7 regulates IL-6 expression by controlling ribonuclease Regnase-1 mRNA (encoded by Zc3h12a gene) stability. Mechanistically, TLR4 activation causes TUT7 to bind directly to the stem-loop structure on Zc3h12a 3′-UTR, thereby promotes Zc3h12a uridylation and degradation. Zc3h12a from LPS-treated TUT7-sufficient macrophages possesses increased oligo-uridylated ends with shorter poly(A) tails, whereas oligo-uridylated Zc3h12a is significantly reduced in Tut7-/- cells after TLR4 activation. Together, our findings reveal the functional role of TUT7 in sculpting TLR4-driven responses by modulating mRNA stability of a selected set of inflammatory mediators.

scholarly journals Identification and characterization of a sequence motif involved in nonsense-mediated mRNA decay.

1995 ◽  
Vol 15 (4) ◽  
pp. 2231-2244 ◽  
Author(s):  
S Zhang ◽  
M J Ruiz-Echevarria ◽  
Y Quan ◽  
S W Peltz
Keyword(s):  
Mrna Decay ◽  
Sequence Motif ◽  
Nonsense Mutations ◽  
Stem Loop ◽  
Cis Acting ◽  
Stem Loop Structure ◽  
Nonsense Codon ◽  
Nonsense Mediated Mrna Decay

In both prokaryotes and eukaryotes, nonsense mutations in a gene can enhance the decay rate or reduce the abundance of the mRNA transcribed from that gene, and we call this process nonsense-mediated mRNA decay. We have been investigating the cis-acting sequences involved in this decay pathway. Previous experiments have demonstrated that, in addition to a nonsense codon, specific sequences 3' of a nonsense mutation, which have been defined as downstream elements, are required for mRNA destabilization. The results presented here identify a sequence motif (TGYYGATGYYYYY, where Y stands for either T or C) that can predict regions in genes that, when positioned 3' of a nonsense codon, promote rapid decay of its mRNA. Sequences harboring two copies of the motif from five regions in the PGK1, ADE3, and HIS4 genes were able to function as downstream elements. In addition, four copies of this motif can function as an independent downstream element. The sequences flanking the motif played a more significant role in modulating its activity when fewer copies of the sequence motif were present. Our results indicate the sequences 5' of the motif can modulate its activity by maintaining a certain distance between the sequence motif and the termination codon. We also suggest that the sequences 3' of the motif modulate the activity of the downstream element by forming RNA secondary structures. Consistent with this view, a stem-loop structure positioned 3' of the sequence motif can enhance the activity of the downstream element. This sequence motif is one of the few elements that have been identified that can predict regions in genes that can be involved in mRNA turnover. The role of these sequences in mRNA decay is discussed.

sRNA STnc150 is involved in virulence regulation of Salmonella Typhimurium by targeting fimA mRNA

2021 ◽  
Vol 368 (18) ◽  
Author(s):  
Jing Li ◽  
Na Li ◽  
Chengcheng Ning ◽  
Yun Guo ◽  
Chunhui Ji ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
Target Genes ◽  
Reporter System ◽  
Stem Loop ◽  
Viral Loads ◽  
Virulence Regulation ◽  
Stem Loop Structure ◽  
Complementary Strain

ABSTRACT Small RNAs (sRNAs) are essential virulent regulators in Salmonella typhimurium (STM). To explore the role of sRNA STnc150 in regulating STM virulence, we constructed a STnc150 deletion strain (ΔSTnc150) and its complementary strain (ΔSTnc150/C). Then, we compared their characteristics to their original parent strain experimentally, identified the target genes of STnc150 and determined the expression levels of target genes. The results showed that the ΔSTnc150 strain exhibited delayed biofilm formation, enhanced adhesion to macrophages, significantly reduced LD50, increased liver and spleen viral loads and more vital pathological damaging ability than its parent and complementary strains. Further, bioinformatics combined with the bacterial dual plasmid reporter system confirmed that the bases 72–88 of STnc150 locating at the secondary stem-loop structure of the STnc150 are complementary with the bases 1–19 in the 5′-terminal of fimA mRNA of the type 1 fimbriae subunit. Western blot analysis showed that fimA protein level was increased in STnc150 strain compared with its parent and complementary strains. Together, this study suggested that STnc150 can down-regulate STM fimA expression at the translation level, which provided insights into the regulatory mechanisms of sRNAs in virulence of STM.

scholarly journals Investigating the role of noncoding regulatory DNA in plasmid development for Yarrowia lipolytica

2020 ◽  
Author(s):  
Carmen Lopez ◽  
Mingfeng Cao ◽  
Zhanyi Yao ◽  
Zengyi Shao
Keyword(s):  
Yarrowia Lipolytica ◽  
Plasmid Stability ◽  
Critical Role ◽  
Fold Increase ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Expression Platform ◽  
Regulatory Dna ◽  
Selection Of

Production of industrially relevant compounds in microbial cell factories can employ either genomes or plasmids as an expression platform. Selection of plasmids as pathway carriers is advantageous for rapid demonstration but poses a challenge of stability. Yarrowia lipolytica has attracted great attention in the past decade for the biosynthesis of chemicals related to fatty acids at titers attractive to industry, and many genetic tools have been developed to explore its oleaginous potential. Our recent studies on the autonomously replicating sequences (ARSs) of nonconventional yeasts revealed that the ARSs from Y. lipolytica showcase a unique structure that includes a previously unannotated sequence (spacer) linking the origin of replication (ORI) and the centromeric (CEN) element and plays a critical role in modulating plasmid behavior. Maintaining a native 645-bp spacer yielded a 4.5-fold increase in gene expression and higher plasmid stability compared to a more universally employed minimized ARS. Testing the modularity of the ARS sub-elements indicated that plasmid stability exhibits a pronounced cargo dependency. Instability caused both plasmid loss and intramolecular rearrangements. Altogether, our work clarifies the appropriate application of various ARSs for the scientific community and sheds light on a previously unexplored DNA element as a potential target for engineering Y. lipolytica.

scholarly journals The conformational landscape of transcription intermediates involved in the regulation of the ZMP-sensing riboswitch from Thermosinus carboxydivorans

2020 ◽  
Vol 48 (12) ◽  
pp. 6970-6979 ◽  
Author(s):  
Oliver Binas ◽  
Tatjana Schamber ◽  
Harald Schwalbe
Keyword(s):  
Ligand Binding ◽  
Conformational Changes ◽  
Single Nucleotide ◽  
Pseudoknot Structure ◽  
Rna Transcripts ◽  
Expression Platform ◽  
Secondary Messengers ◽  
Conformational Landscape ◽  
Response To Change ◽  
Aptamer Domain

Abstract Recently, prokaryotic riboswitches have been identified that regulate transcription in response to change of the concentration of secondary messengers. The ZMP (5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR))-sensing riboswitch from Thermosinus carboxydivorans is a transcriptional ON-switch that is involved in purine and carbon-1 metabolic cycles. Its aptamer domain includes the pfl motif, which features a pseudoknot, impeding rho-independent terminator formation upon stabilization by ZMP interaction. We herein investigate the conformational landscape of transcriptional intermediates including the expression platform of this riboswitch and characterize the formation and unfolding of the important pseudoknot structure in the context of increasing length of RNA transcripts. NMR spectroscopic data show that even surprisingly short pre-terminator stems are able to disrupt ligand binding and thus metabolite sensing. We further show that the pseudoknot structure, a prerequisite for ligand binding, is preformed in transcription intermediates up to a certain length. Our results describe the conformational changes of 13 transcription intermediates of increasing length to delineate the change in structure as mRNA is elongated during transcription. We thus determine the length of the key transcription intermediate to which addition of a single nucleotide leads to a drastic drop in ZMP affinity.

scholarly journals An RNA Pseudoknot in the 3′ End of the Arterivirus Genome Has a Critical Role in Regulating Viral RNA Synthesis

2007 ◽  
Vol 81 (17) ◽  
pp. 9426-9436 ◽  
Author(s):  
Nancy Beerens ◽  
Eric J. Snijder
Keyword(s):  
Rna Synthesis ◽  
Critical Role ◽  
Equine Arteritis Virus ◽  
Viral Rna ◽  
Replicase Gene ◽  
Loop Structure ◽  
Minus Strand ◽  
Stem Loop ◽  
Loop Region ◽  
Stem Loop Structure

ABSTRACT In the life cycle of plus-strand RNA viruses, the genome initially serves as the template for both translation of the viral replicase gene and synthesis of minus-strand RNA and is ultimately packaged into progeny virions. These various processes must be properly balanced to ensure efficient viral proliferation. To achieve this, higher-order RNA structures near the termini of a variety of RNA virus genomes are thought to play a key role in regulating the specificity and efficiency of viral RNA synthesis. In this study, we have analyzed the signals for minus-strand RNA synthesis in the prototype of the arterivirus family, equine arteritis virus (EAV). Using site-directed mutagenesis and an EAV reverse genetics system, we have demonstrated that a stem-loop structure near the 3′ terminus of the EAV genome is required for RNA synthesis. We have also obtained evidence for an essential pseudoknot interaction between the loop region of this stem-loop structure and an upstream hairpin residing in the gene encoding the nucleocapsid protein. We propose that the formation of this pseudoknot interaction may constitute a molecular switch that could regulate the specificity or timing of viral RNA synthesis. This hypothesis is supported by the fact that phylogenetic analysis predicted the formation of similar pseudoknot interactions near the 3′ end of all known arterivirus genomes, suggesting that this interaction has been conserved in evolution.

The critical role of dimer formation in monosaccharides binding to human serum albumin

2018 ◽  
Vol 20 (5) ◽  
pp. 3249-3257 ◽  
Author(s):  
Prapasiri Pongprayoon ◽  
Toshifumi Mori
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Critical Role ◽  
Binding Pocket ◽  
Dimer Formation ◽  
Dimeric Structure

Monosaccharides are found to bind tightly to human serum albumin when a dimeric structure is formed in the binding pocket.

scholarly journals Correction to “Critical Role of the Secondary Binding Pocket in Modulating the Enzymatic Activity of DUSP5 toward Phosphorylated ERKs”

Biochemistry ◽  
2018 ◽  
Vol 57 (26) ◽  
pp. 3987-3987
Author(s):  
Marat R. Talipov ◽  
Jaladhi Nayak ◽  
Michael Lepley ◽  
Robert D. Bongard ◽  
Daniel S. Sem ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Critical Role ◽  
Binding Pocket
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