Recombinant Gracilaria tenuistipitata var. liui 30S ribosomal protein S11, chloroplastic (rps11)

What is the genetic structure and location of the rps11 gene in the chloroplast genome of Gracilaria tenuistipitata var. liui?

To identify and characterize the rps11 gene structure:

• Extract total genomic DNA using established protocols, such as QIAGEN DNEasy Plant Mini Kit

• Sequence using next-generation sequencing platforms (e.g., Illumina)

• Perform de novo assembly and annotation using tools like DOGMA with an e-value cutoff of 10^-20 for BLAST hits

• Manually check alignments and annotate the corresponding ORF in the genome sequence

How does the rps11 gene in Gracilaria tenuistipitata var. liui differ from other red algal species?

Comparative analysis of rps11 across red algal species reveals notable differences. For instance, the rps11 gene in Ludwigia octovalvis (Lo) is shorter (345 bp) compared to the same gene in Ludwigia grandiflora (Lgh) and Ludwigia peploides (Lpm) (417 bp). This difference is due to a frameshift mutation in Lo caused by the deletion of a nucleotide at position 311 .

When comparing rps11 across various red algal species, researchers have documented the following differences:

To investigate these differences:

• Extract chloroplast DNA

• Amplify the rps11 gene using PCR with specific primers

• Sequence the gene from multiple individuals of each species

• Perform multiple sequence alignment using tools like MAFFT v.7

• Calculate genetic distance using models such as the Kimura 2-parameter model

What bioinformatic tools and protocols are recommended for identification and annotation of the rps11 gene in newly sequenced red algal chloroplast genomes?

For accurate identification and annotation of the rps11 gene in newly sequenced red algal chloroplast genomes, researchers should implement the following workflow:

• Initial Genome Assembly and Quality Control:

  • Use hybrid assembly approaches combining both long and short reads for optimal results

  • For short read sequencing: perform quality-trimming (error threshold = 0.05, n ambiguities = 2) using tools like CLC Genomics Workbench

  • For long reads: utilize platforms such as Oxford Nanopore or PacBio

  • Hybrid assembly approaches harness the accuracy of short reads and the length advantages of long reads, as demonstrated in studies of chloroplasts in Eucalyptus, Falcataria, Carex, and Cypripedium

• Chloroplast Genome Annotation:

  • Submit assembled genome to GeSeq for preliminary annotation

  • Use DOGMA with an e-value cutoff of 10^-20 for BLAST hits

  • Check alignments manually and annotate the corresponding ORF

• Specific rps11 Identification:

  • Extract known rps11 sequences from reference genomes (e.g., Gracilaria tenuistipitata var. liui)

  • Use these sequences as queries in BLAST searches against the new genome

  • Verify gene boundaries by examining start and stop codons (considering ATG, GTG, and TTG as potential start codons)

• Functional Domain Analysis:

  • Translate identified ORFs using the standard genetic code

  • Submit translations to phmmer, searching against the UniProtKB database

  • Use the InterProScan to identify functional domains

• Verification and Manual Curation:

  • Confirm gene identity through reciprocal BLAST

  • Check for potential frameshift mutations that might affect gene length

  • Examine gene context within the ribosomal supercluster

This comprehensive approach ensures accurate identification and characterization of the rps11 gene in newly sequenced chloroplast genomes.

What experimental approaches can be used to study the function of chloroplastic rps11 in translation?

To investigate the function of chloroplastic rps11 in translation, researchers can employ several complementary experimental approaches:

• In vitro Reconstitution Assays:

  • Express recombinant rps11 protein and other ribosomal components

  • Reconstitute 30S subunits following established protocols where proteins are added in groups

  • Group I (assembly initiator proteins): S4, S7, S8, S15, S17, S20

  • Group II: S5, S6, S9, S11, S12, S13, S16, S18, S19

  • Group III: S2, S3, S10, S14, S21

  • Assess reconstitution efficiency by measuring sedimentation profiles and functional assays such as tRNA binding and polyphenylalanine synthesis

• Targeted Mutagenesis:

  • Introduce specific mutations in the rps11 gene based on alignments with other species

  • Express mutant proteins and assess their ability to incorporate into ribosomes

  • Compare translation efficiency of wild-type and mutant rps11-containing ribosomes

• Protein-RNA Interaction Studies:

  • Perform RNA immunoprecipitation to identify RNA partners of rps11

  • Use chemical crosslinking and structural probing to map interaction sites

  • Employ dimethyl sulfate modification patterns to assess 16S ribosomal RNA in reconstituted 30S subunits

• Comparative Analysis of Frameshift Mutations:

  • Create constructs mimicking the frameshift mutation observed in Ludwigia octovalvis

  • Express both wild-type and frameshift mutant proteins

  • Compare structural stability and functional activity of both variants

  • Assess potential consequences for ribosome assembly and translation fidelity

These methods collectively provide a comprehensive approach to understanding rps11's role in chloroplastic translation.

What evidence exists for horizontal or endosymbiotic gene transfer of rps11 in red algae?

Numerous studies have documented evidence for endosymbiotic gene transfer (EGT) of ribosomal protein genes, including rps11, in red algae. This process involves the transfer of genes from the chloroplast genome to the nuclear genome during evolution.

The evidence for EGT of rps11 includes:

• Genomic Distribution Patterns:

  • rps11 and other ribosomal protein genes have been identified in nuclear genomes of multiple red algal species

  • These genes are scattered throughout the nuclear genome, suggesting that either independent gene transfers occurred or that post-EGT translocations separated the genes that were originally transferred as an operon

• Conserved Gene Clusters:

  • In the nine red algal species investigated in a comprehensive study, researchers identified nuclear copies of rpl6, rpl14, rpl16, rps11, rps12, and rps14

  • Some of these ribosomal subunit genes remain clustered, preserving their ancestral organization

• Frameshift Mutations in Chloroplast Copies:

  • The presence of frameshift mutations in chloroplast copies of rps11 (as seen in Ludwigia octovalvis) may indicate relaxed selection pressure following gene transfer to the nucleus

  • Such rps11 horizontal transfers have been reported specifically in the mitochondrial genomes of various plant families

• Evidence from Comparison Studies:

  • Phylogenetic analyses of rps11 sequences from chloroplasts, mitochondria, and nuclear genomes show patterns consistent with ancient gene transfer events

  • Protein targeting sequences attached to nuclear-encoded copies indicate adaptation for re-import into organelles

Understanding these transfer events is crucial for comprehending the evolution of chloroplast genomes and the functional compartmentalization of the translation machinery in red algae.

What are the known consequences of frameshift mutations in the rps11 gene on protein function and ribosome assembly?

• Altered Protein Structure and Function:

  • The frameshift mutation in Ludwigia octovalvis results in a shorter protein (345 bp vs. 417 bp), potentially affecting its tertiary structure

  • Such mutations may alter binding interfaces crucial for interaction with 16S rRNA and other ribosomal proteins

  • Functional domains necessary for translation accuracy may be disrupted

• Impact on Ribosome Assembly:

  • Studies with E. coli ribosomal proteins suggest that S11 is part of Group II proteins in the assembly pathway

  • Defects in S11 could disrupt the ordered assembly process, potentially resulting in incomplete or non-functional ribosomes

  • As demonstrated in reconstitution experiments, the sequential addition of proteins is critical for proper ribosome assembly, and mutations in assembly components can compromise this process

• Translational Consequences:

  • S11 plays an important role in translational accuracy

  • Mutations may affect the recognition of mRNA codons and tRNA anticodons, potentially leading to mistranslation

  • The ribosomal ambiguity (ram) phenotype observed with some ribosomal protein mutations may result from frameshift mutations in S11 or related proteins

• Evolutionary Implications:

  • The persistence of frameshift mutations in chloroplast rps11 genes suggests potential compensation mechanisms

  • Possible scenarios include:
    a) Gene transfer to the nucleus with subsequent loss of function in the chloroplast copy
    b) Functional redundancy within the translation machinery
    c) Translation reinitiation downstream of the frameshift

  • Functional analysis is necessary to determine whether truncated proteins have retained some activity or have completely lost function

Understanding these consequences requires careful functional characterization through reconstitution experiments and comparative analyses of translation efficiency in systems with wild-type versus mutant S11 proteins.

How can recombinant rps11 be used in reconstitution experiments to study chloroplast ribosome assembly and function?

Reconstitution experiments using recombinant rps11 provide powerful insights into chloroplast ribosome assembly and function. Based on established protocols with bacterial ribosomes, researchers can adapt the following methodological framework specifically for chloroplastic ribosomes:

• Expression and Purification of Recombinant Components:

  • Clone the rps11 gene from Gracilaria tenuistipitata var. liui into an appropriate expression vector

  • Express the protein with a purification tag (e.g., His-tag) in a suitable host system

  • Purify using affinity chromatography followed by size exclusion chromatography to ensure high purity (>90%)

  • Similarly express and purify all other chloroplast ribosomal proteins required for 30S subunit assembly

• Sequential Assembly Protocol:

  • Based on the established E. coli 30S reconstitution protocols, organize chloroplast ribosomal proteins into functional groups:

    • Group I (primary binding proteins): Include S4, S7, S8, S15, S17, and S20

    • Group II (secondary binding proteins): Include S5, S6, S9, S11, S12, S13, S16, S18, and S19

    • Group III (tertiary binding proteins): Include S2, S3, S10, S14, and S21

  • Add these groups sequentially to purified chloroplast 16S rRNA with incubation periods at 42°C

  • This sequential approach significantly improves reconstitution efficiency compared to single-step methods

• Functional Validation Assays:

  • Verify proper assembly by analyzing sedimentation profiles on sucrose gradients

  • Assess tRNA binding capacity in a template-dependent manner

  • Test the ability of reconstituted 30S subunits to associate with 50S subunits to form 70S ribosomes

  • Measure translation activity using poly(U)-directed polyphenylalanine synthesis assays

• Structure-Function Analysis:

  • Create variant forms of rps11 (point mutations, deletions, or chimeric proteins)

  • Incorporate these variants into the reconstitution protocol

  • Compare assembly efficiency and functional parameters of ribosomes containing wild-type versus variant rps11

  • Use these data to map functional domains and critical residues

• RNA-Protein Interaction Mapping:

  • Employ dimethyl sulfate modification patterns of 16S rRNA to assess structural changes induced by rps11 binding

  • Compare modification patterns between natural 30S subunits and those reconstituted with recombinant proteins

  • Identify specific nucleotides protected by or exposed due to rps11 interaction

This comprehensive approach allows researchers to dissect the specific contribution of rps11 to ribosome assembly and function while providing a platform for investigating the effects of naturally occurring mutations or designed variants.

What structural and functional relationships exist between rps11 and other components of the chloroplast translation machinery?

The structural and functional relationships between rps11 and other components of the chloroplast translation machinery are complex and multifaceted. Based on research findings, these relationships can be categorized as follows:

• Interactions within the Ribosomal Supercluster:

  • The rps11 gene is located within a highly conserved ribosomal supercluster in the red algal plastid lineage

  • This supercluster contains approximately 29 genes, and the relative positioning suggests co-regulation and functional relationships

  • Three genes in this cluster (rps11, rps13, and rpl31) show inversion events in some species, indicating evolutionary plasticity while maintaining functional proximity

• Protein-Protein Interactions in the 30S Subunit:

  • Based on bacterial ribosome studies, S11 is known to interact directly with several other ribosomal proteins:

    • Primary interactions with S7, S18, and S21

    • Secondary interactions with S5 and S12

  • These interactions are critical for proper assembly of the head domain of the 30S subunit

  • S11, S5, and S12 form a functional unit that plays an important role in translational accuracy

• Protein-RNA Interactions:

  • S11 binds directly to specific regions of the 16S rRNA

  • These interactions help stabilize the tertiary structure of the rRNA

  • The position of S11 on the 30S subunit places it near the decoding center, consistent with its role in translational accuracy

• Functional Relationships in Translation:

  • S11 participates in mRNA unwinding by the ribosome, possibly by forming part of a processivity clamp

  • Works with S5 and S12 to ensure translational accuracy

  • Many suppressors of streptomycin-dependent mutants of protein S12 are found in S11

  • Some mutations in S11 decrease translational accuracy, resulting in ribosomal ambiguity (ram) mutations

• Coordinated Gene Expression:

  • In bacterial systems, S11 functions as a translational repressor protein

  • It controls the translation of the alpha-operon (which encodes S13, S11, S4, RNA polymerase alpha subunit, and L17) by binding to its mRNA

  • Whether similar autoregulatory mechanisms exist in chloroplasts remains to be fully elucidated

These relationships highlight the integrated nature of S11's function within the ribosome and suggest that mutations or alterations in this protein could have far-reaching effects on translation and chloroplast function.

How can CRISPR-based technologies be applied to study rps11 function in red algal chloroplasts?

CRISPR-based technologies offer unprecedented opportunities for studying gene function in vivo, but applying these techniques to chloroplast genes in red algae presents unique challenges and opportunities. Here's a comprehensive methodological approach for using CRISPR to study rps11 function in red algal chloroplasts:

• Selection of CRISPR System Components:

  • Choose CRISPR-Cas9 or CRISPR-Cpf1 systems based on PAM requirements and editing efficiency

  • Design guide RNAs (gRNAs) specific to the rps11 gene sequence, avoiding regions with potential off-target sites

  • Consider using a ribonucleoprotein (RNP) complex approach (pre-assembled Cas protein and gRNA) to enhance delivery efficiency and reduce off-target effects

• Delivery Methods for Red Algae:

  • Optimize protoplast isolation from Gracilaria tenuistipitata var. liui using enzymatic digestion of cell walls

  • Employ electroporation for direct delivery of CRISPR components to protoplasts

  • Alternatively, use biolistic transformation (gene gun) for delivery to intact cells

  • Consider microinjection for targeted delivery to specific cells or chloroplasts

• Design of Editing Strategies:

  • Knockout studies: Target the coding region of rps11 to create frameshift mutations or premature stop codons

  • Base editing: Use cytidine or adenine base editors for precise point mutations without double-strand breaks

  • Prime editing: Design pegRNAs to introduce specific mutations, insertions, or deletions with high precision

  • HDR-based editing: Supply repair templates to introduce specific mutations or reporter genes

• Validation and Phenotypic Analysis:

  • Screen transformants using PCR and sequencing to identify successful editing events

  • Perform Western blotting to verify changes at the protein level

  • Assess chloroplast ribosome assembly and function through polysome profiling

  • Measure chloroplast translation rates using radioactive amino acid incorporation assays

  • Analyze growth rates and physiological parameters under various conditions

• Methods to Overcome Unique Challenges:

  • Polyploidy of chloroplast genomes: Extend culture time to allow complete segregation of transformed genomes

  • Multiple chloroplasts per cell: Use selection markers or screening methods to identify cells with homoplasmic edits

  • Off-target effects: Thoroughly validate edited lines by whole-genome sequencing

  • Low transformation efficiency: Optimize culture conditions for protoplast regeneration and growth after transformation

• Complementation Strategies:

  • Design rescue experiments by introducing wild-type or variant rps11 genes

  • Consider expressing nuclear-encoded, chloroplast-targeted S11 protein to complement chloroplast knockout

  • Use inducible promoters to control expression of complementing genes for temporal studies

This comprehensive approach provides a framework for applying CRISPR technologies to study rps11 function in red algal chloroplasts, contributing valuable insights into chloroplast ribosome assembly, translation, and the evolutionary significance of ribosomal proteins in these organisms.

What are the most effective protocols for expressing and purifying recombinant chloroplastic rps11 protein from Gracilaria tenuistipitata var. liui?

Expressing and purifying recombinant chloroplastic rps11 protein from Gracilaria tenuistipitata var. liui requires careful optimization of multiple parameters. Based on established methods for similar ribosomal proteins, the following comprehensive protocol is recommended:

• Gene Cloning and Vector Construction:

  • Amplify the rps11 coding sequence from G. tenuistipitata var. liui chloroplast DNA using PCR

  • Optimize codon usage for the expression host if necessary

  • Clone into an expression vector with an appropriate fusion tag (His-tag is commonly used for ribosomal proteins)

  • Consider using vectors with tightly controlled promoters (T7 or similar) to minimize potential toxicity

• Expression System Selection:

  • Bacterial expression (E. coli):

    • Use BL21(DE3) or similar strains optimized for protein expression

    • Consider Rosetta or CodonPlus strains if codon bias is an issue

    • For challenging expressions, try specialized strains like C41(DE3) designed for potentially toxic proteins

  • Eukaryotic alternatives:

    • Baculovirus expression in insect cells may provide better folding for complex proteins

    • Yeast expression systems can be considered for proteins requiring eukaryotic post-translational modifications

• Optimization of Expression Conditions:

  • Perform small-scale expression trials varying:

    • Induction temperature (15-37°C)

    • IPTG concentration (0.1-1.0 mM)

    • Induction duration (2-24 hours)

    • Media composition (LB, TB, or defined media)

  • Analyze results by SDS-PAGE to determine optimal conditions

  • For problematic expressions, consider auto-induction media or co-expression with chaperones

• Protein Purification Strategy:

  • Cell Lysis:

    • Use sonication or French press for efficient bacterial cell disruption

    • Include protease inhibitors in all buffers

    • Optimize salt concentration in lysis buffer to enhance solubility (typically 300-500 mM NaCl)

  • Affinity Chromatography:

    • For His-tagged rps11, use Ni-NTA resin

    • Wash extensively to remove non-specifically bound proteins

    • Include a low concentration of imidazole (10-20 mM) in wash buffers

    • Elute with an imidazole gradient or step elution

  • Secondary Purification:

    • Employ ion exchange chromatography as a second step

    • Consider size exclusion chromatography for final polishing

    • Aim for >90% purity for functional studies

• Assessment of Protein Quality:

  • Verify purity by SDS-PAGE

  • Confirm identity by Western blotting and/or mass spectrometry

  • Assess protein folding by circular dichroism spectroscopy

  • Evaluate RNA binding activity using electrophoretic mobility shift assays with 16S rRNA fragments

• Optimization for Specific Applications:

  • For structural studies:

    • Further purification may be necessary

    • Consider tag removal using specific proteases

    • Perform buffer screening to identify conditions promoting stability

  • For functional reconstitution:

    • Verify activity in ribosome reconstitution assays

    • Assess proper incorporation into 30S subunits

    • Test functionality in in vitro translation systems

By following this detailed protocol and optimizing each step for the specific characteristics of rps11, researchers can obtain high-quality recombinant protein suitable for structural and functional studies.

How can RNA-protein interaction studies be designed to investigate the binding partners and functional domains of chloroplastic rps11?

Investigating RNA-protein interactions involving chloroplastic rps11 requires a multi-faceted approach combining in vitro and, where possible, in vivo methods. The following comprehensive experimental design provides a methodological framework for such studies:

• In Vitro Binding Assays:

  • Electrophoretic Mobility Shift Assays (EMSA):

    • Generate recombinant rps11 protein following established purification protocols

    • Prepare labeled RNA fragments (either radioactive or fluorescent) representing potential binding regions of 16S rRNA

    • Incubate protein and RNA under various buffer conditions and analyze complex formation by native gel electrophoresis

    • Determine binding affinity constants and specificity through titration experiments

  • Filter Binding Assays:

    • Use nitrocellulose filters to capture protein-RNA complexes

    • Apply labeled RNA and protein mixtures to filters and wash to remove unbound RNA

    • Quantify bound RNA to determine binding constants with greater precision than EMSA

  • Surface Plasmon Resonance (SPR):

    • Immobilize either rps11 or RNA target on a sensor chip

    • Monitor real-time binding kinetics of the interaction

    • Determine kon and koff rates as well as binding affinity

• Structural Mapping of Interaction Sites:

  • RNA Structure Probing:

    • Employ chemical probing methods (DMS, SHAPE) to identify RNA nucleotides protected by rps11 binding

    • Use ribonucleases with different specificities to map protein footprints on target RNAs

    • Compare modification patterns between naked RNA and RNA-protein complexes

  • UV Crosslinking and Immunoprecipitation:

    • Expose rps11-RNA complexes to UV light to induce covalent bonds at contact points

    • Digest away unprotected RNA and sequence remaining fragments to identify binding sites

    • For more precise mapping, consider PAR-CLIP or similar advanced crosslinking techniques

• Mutational Analysis to Define Functional Domains:

  • Protein Domain Mapping:

    • Generate truncation variants of rps11 to identify minimal binding domains

    • Create point mutations in conserved residues identified through sequence alignment

    • Test each variant's binding capacity using the assays described above

  • RNA Target Mutagenesis:

    • Introduce mutations in the putative binding sites of target RNAs

    • Assess how these mutations affect binding affinity and specificity

    • Create compensatory mutations to restore interactions disrupted by initial mutations

• Reconstitution Experiments:

  • Incorporate wild-type and mutant rps11 proteins into 30S ribosomal subunit reconstitution assays

  • Assess the impact of mutations on ribosome assembly efficiency

  • Determine functional consequences through translation activity measurements

  • Use dimethyl sulfate modification patterns to assess structural integrity of reconstituted subunits

• Advanced Structural Approaches:

  • NMR Spectroscopy:

    • For smaller RNA-protein complexes, use solution NMR to obtain atomic-resolution data

    • Map chemical shift perturbations upon complex formation to identify interaction surfaces

  • Cryo-Electron Microscopy:

    • For larger complexes or full ribosomal assemblies, use cryo-EM for structural determination

    • Compare structures with and without rps11 to identify conformational changes

  • X-ray Crystallography:

    • Attempt crystallization of rps11 alone and in complex with RNA targets

    • Solve high-resolution structures to identify specific interaction details