Recombinant Synechocystis sp. Uncharacterized RNA pseudouridine synthase slr1629 (slr1629)
Description
Introduction to Synechocystis sp. RNA Pseudouridine Synthase slr1629
The Synechocystis sp. PCC6803 genome encodes two photolyase-like genes, slr0854 and sll1629. While slr0854 exhibits classical photolyase activity, sll1629 has been identified as a putative RNA pseudouridine synthase with structural similarities to cryptochromes rather than canonical pseudouridine synthases . Despite initial annotations suggesting pseudouridylation activity, functional studies reveal distinct characteristics, positioning sll1629 within the cryptochrome family .
Genomic Context
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Chromosomal location: sll1629 is located on the chromosome of Synechocystis sp. PCC6803, distinct from the slr0854 photolyase gene .
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Sequence homology: Shares 35–40% similarity with eukaryotic cryptochromes and photolyases but lacks conserved residues critical for photolyase activity .
Phylogenetic Analysis
A phylogenetic tree (Figure 1) places sll1629 closer to cryptochromes than photolyases or pseudouridine synthases, suggesting divergent evolution from typical RNA-modifying enzymes .
| Protein | Closest Homolog | Key Functional Residues | Activity |
|---|---|---|---|
| slr0854 | CPD photolyase | Catalytic FAD-binding site | DNA repair (CPD-specific) |
| sll1629 | Cryptochromes | Absent photolyase motifs | No detectable DNA repair |
Enzyme Activity Assays
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Substrate specificity: Recombinant slr1629 protein purified from E. coli showed no affinity for cyclobutane pyrimidine dimers (CPDs) or RNA substrates in vitro .
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Catalytic activity: Unlike pseudouridine synthases (e.g., human PUS1 or yeast Pus4), slr1629 lacks the conserved aspartate residue required for uridine isomerization .
UV Sensitivity and Phenotypic Analysis
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Mutant studies: Disruption of sll1629 in Synechocystis had no significant impact on growth under UV stress, unlike slr0854 mutants .
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RNA-binding assays: Band-shift experiments revealed no RNA-binding activity, further distinguishing it from pseudouridine synthases .
Domain Architecture
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Photolyase/cryptochrome domain: Contains a flavin-binding region but lacks the RNA-interacting motifs found in pseudouridine synthases .
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Absence of catalytic pocket: Structural predictions indicate no base-flipping mechanism critical for pseudouridylation .
Evolutionary Implications
The sll1629 gene represents a bacterial cryptochrome lineage, diverging early from photolyases and pseudouridine synthases. Its lack of RNA-modifying activity challenges initial annotations, highlighting the need for re-evaluation of "uncharacterized" enzymes in microbial genomes .
Research Gaps and Future Directions
Product Specs
Q&A
Given the limited specific information available on the recombinant Synechocystis sp. uncharacterized RNA pseudouridine synthase slr1629 (slr1629), we will focus on general aspects of pseudouridine synthases and their research applications. Here is a collection of FAQs tailored for researchers:
Q: What are pseudouridine synthases, and how do they function?
A: Pseudouridine synthases are enzymes responsible for converting uridine residues to pseudouridine in RNA. They play a crucial role in RNA modification, affecting RNA stability and interactions with proteins. The mechanism involves a dissociation-rebound process where the uracil detaches from the ribose and then rebinds to form pseudouridine .
Q: How do pseudouridine synthases recognize their target RNAs?
A: Recognition often involves specific structural motifs within the RNA and interactions with the enzyme's active site. For example, human PUS7 recognizes RNA through its extended RNA binding surface and structural features of the target RNA .
Q: What methods can be used to study pseudouridine synthase activity?
A: Techniques include in vitro assays like tritium release assays to measure pseudouridylation efficiency. Additionally, high-throughput sequencing and computational predictions can identify potential targets and structural motifs required for modification .
Q: How can researchers analyze pseudouridine residues in RNA?
A: Methods such as CMCT (1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate) treatment followed by reverse transcription can identify pseudouridine residues in RNA .
Q: How do researchers handle contradictory data in pseudouridine synthase studies?
A: Contradictory data can arise from differences in experimental conditions or RNA substrates. Researchers should carefully analyze assay conditions, RNA structures, and enzyme concentrations to reconcile discrepancies. Additionally, using multiple experimental approaches can help validate findings .
Q: What role does bioinformatics play in predicting pseudouridine synthase targets?
A: Bioinformatics tools are crucial for predicting potential pseudouridylation sites based on RNA structure and sequence motifs. These predictions can guide experimental validation and expand the understanding of pseudouridine synthase specificity .
Q: How might pseudouridine synthases be used in RNA editing and therapeutics?
A: Pseudouridine synthases, particularly those like box H/ACA, could be engineered to target specific RNAs for modification, potentially correcting genetic lesions or altering gene expression. This could involve designing guide RNAs to direct pseudouridylation to specific sites .
Q: What are the implications of pseudouridine synthase activity on RNA function and disease?
A: Pseudouridylation affects RNA stability and interactions, which can influence gene expression and cellular function. Dysregulation of pseudouridine synthases has been linked to diseases such as dyskeratosis congenita and cancer .
Q: What are some future research directions for pseudouridine synthases?
A: Future studies should focus on elucidating the structural basis of enzyme-substrate interactions, exploring the physiological roles of pseudouridine in different RNAs, and developing therapeutic applications based on pseudouridine synthase activity .
Q: How can researchers leverage pseudouridine synthases for novel RNA modifications?
A: By understanding the specificity and mechanisms of pseudouridine synthases, researchers can engineer these enzymes to introduce modifications at specific sites, potentially expanding their therapeutic potential .
