Recombinant Enterococcus faecalis Ribonuclease M5 (rnmV)
Description
Introduction to Recombinant Enterococcus faecalis Ribonuclease M5 (rnmV)
Recombinant Enterococcus faecalis Ribonuclease M5, denoted as rnmV, is an enzyme derived from the bacterium Enterococcus faecalis. This ribonuclease plays a crucial role in RNA processing, particularly in the maturation of ribosomal RNA (rRNA). The enzyme is significant for its ability to cleave RNA molecules, facilitating the correct processing of precursor rRNA into functional forms necessary for protein synthesis.
Role in rRNA Processing
Ribonuclease M5 is essential for the proper processing of both the 5' and 3' ends of the 5S rRNA precursor. It cleaves double-stranded regions within the precursor, yielding mature 5S rRNA, which is a vital component of the ribosomal structure. This process is critical for maintaining the integrity and functionality of ribosomes in bacterial cells .
Enzymatic Activity
The enzymatic activity of rnmV includes:
-
Endonucleolytic cleavage: It specifically targets double-stranded RNA regions.
-
Maturation of rRNA: Ensures that the ribosomal RNA components are correctly processed for assembly into ribosomes.
Expression Systems
Recombinant technology has enabled the production of rnmV in various expression systems, including:
-
Escherichia coli: Commonly used due to its rapid growth and well-established genetic tools.
-
Yeast systems: Sometimes preferred for post-translational modifications.
Purification Techniques
The purification of recombinant rnmV typically involves:
-
Affinity chromatography: Utilizes tags such as His-tags for efficient isolation.
-
Ion exchange chromatography: Further refines purity based on charge properties.
Functional Studies
Functional assays have demonstrated that:
-
Mutations in critical residues can significantly impair enzymatic activity.
-
The enzyme's activity can be influenced by environmental factors such as pH and temperature.
Potential Uses
Recombinant Enterococcus faecalis Ribonuclease M5 holds promise in various biotechnological applications:
-
Molecular biology: Used as a tool for RNA manipulation and analysis.
-
Therapeutics: Potential development as an antibacterial agent targeting specific pathogens.
Comparative Analysis with Other Ribonucleases
| Feature | Ribonuclease M5 | Other Ribonucleases |
|---|---|---|
| Source | Enterococcus faecalis | Various (e.g., E. coli, Bacillus subtilis) |
| Specificity | Double-stranded RNA | Varies by type |
| Application | RNA processing | RNA degradation, therapeutic uses |
| Recombinant Production Ease | Moderate | Varies widely |
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for customized fulfillment.
Note: All proteins are shipped with standard blue ice packs. Dry ice shipping requires advance notice and incurs additional charges.
The tag type is determined during the production process. If a specific tag type is required, please inform us for preferential development.
Target Background
Essential for proper processing of both 5' and 3' ends of the 5S rRNA precursor. It cleaves both sides of a double-stranded region, generating mature 5S rRNA in a single step.
KEGG: efa:EF0935
STRING: 226185.EF0935
Q&A
Basic Research Questions
-
What is Ribonuclease M5 (rnmV) in Enterococcus faecalis and what is its primary function?
Ribonuclease M5 (RNase M5) in E. faecalis is an endoribonuclease involved in RNA processing, particularly in 5S rRNA maturation. Similar to its homolog in Bacillus subtilis, it catalyzes the removal of nucleotides from both the 5'- and 3'-termini of precursor 5S rRNA.
RNase M5 performs a double-stranded RNA cleavage reaction, removing extensions from pre-5S rRNA molecules to generate mature 5S rRNA. This enzyme contains two key domains: an N-terminal catalytic Toprim domain and a C-terminal RNA-binding domain. Unlike RNase E in E. coli which processes both rRNA and mRNA, RNase M5 appears to be highly specific for 5S rRNA processing .
-
How is RNase M5 activity regulated in E. faecalis?
RNase M5 activity requires the presence of cofactors, primarily the ribosomal protein L18, which acts as an RNA chaperone. L18 binds to 5S rRNA and presents the substrate to RNase M5 in the correct conformation for cleavage .
The enzyme utilizes a two-metal-ion catalytic mechanism, with two Mg²⁺ ions in the active site pocket of the catalytic Toprim domain that are essential for catalysis. These metal ions are coordinated by conserved acidic residues including E10, D14, D56, D58, and E96 .
Research suggests that unlike some other ribonucleases that have broader substrate preferences, RNase M5 activity appears to be tightly controlled and specific to 5S rRNA processing, with no evidence of direct mRNA processing activity .
-
What is the structural composition of E. faecalis RNase M5?
RNase M5 consists of two distinct domains connected by a flexible linker:
-
N-terminal Toprim domain (NTD): Spans residues 1-113 and contains the catalytic site with five conserved acidic residues (E10, D14, D56, D58, and E96) that coordinate two Mg²⁺ ions essential for catalysis. The NTD shows a central β-sheet surrounded by four α-helices (α1-α4) and includes a long loop with two small α-helical segments.
-
C-terminal domain (CTD): Spans residues 123-187 and forms an RNA-binding domain with four α-helices (α5-α8). It has a novel RNA-binding fold that resembles a death domain fold but functions differently.
-
Linker region: Residues 114-122 form a flexible connection between the two domains .
This structure enables RNase M5 to bind as a monomer to the 50S ribosomal subunit, with the NTD positioned at the pre-5S rRNA 3'-cleavage site and the CTD driving binding to the ribonucleoprotein substrate .
-
-
How does RNase M5 differ from other ribonucleases in E. faecalis?
E. faecalis contains several ribonucleases with distinct functions:
Ribonuclease Gene Function Essentiality Stress Response RNase M5 rnmV 5S rRNA maturation Unknown in E. faecalis Unknown RNase J1 rnjA RNA turnover, 5' to 3' exonuclease Essential Unknown RNase J2 rnjB RNA turnover, 5' to 3' exonuclease Dispensable Cold, oxidative, bile salts stress RNase III rnc dsRNA cleavage, rRNA/tRNA processing Dispensable Cold, oxidative, bile salts stress RNase Y rny Initial mRNA decay, endonuclease Dispensable General fitness Unlike RNases J1/J2, Y, and III that process various RNA species and affect stress responses and virulence, RNase M5 appears to have specialized functions primarily in rRNA processing. While other RNases like RNase E in E. coli have numerous mRNA substrates, RNase M5 seems to be highly specific for 5S rRNA processing .
-
Why is the cofactor L18 essential for RNase M5 function?
The ribosomal protein L18 serves as an essential cofactor for RNase M5 activity through several mechanisms:
-
RNA chaperone function: L18 binds to pre-5S rRNA and molds it into an optimal conformation for recognition by RNase M5. This conformational arrangement is crucial for proper substrate presentation.
-
Substrate preparation: By binding to pre-5S rRNA, L18 positions the rRNA precursor extensions in proximity to the NTD of RNase M5 for efficient cleavage.
-
Substrate specificity: The requirement for L18 binding ensures that only properly formed ribonucleoprotein complexes containing pre-5S rRNA are processed, preventing nonspecific RNA cleavage.
In B. subtilis, experiments have shown that without L18, RNase M5 shows minimal to no activity on pre-5S rRNA substrates. This dependency on L18 differentiates RNase M5 from many other ribonucleases and contributes to its high substrate specificity .
-
