Gene: rpsD (PA4239)
Protein: Small ribosomal subunit protein uS4 (30S ribosomal protein S4)
UniProt: O52759
Organism: Pseudomonas aeruginosa PAO1
The rpsD gene of P. aeruginosa PAO1 was identified through the complete genome sequencing project [PMID:10984043, "Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen"]. The gene is designated PA4239 in the PAO1 genome.
uS4 is a primary rRNA-binding protein of the bacterial 30S ribosomal subunit. According to PubMed, the UniProt record (HAMAP-Rule:MF_01306) states: "One of the primary rRNA binding proteins, it binds directly to 16S rRNA where it nucleates assembly of the body of the 30S subunit." It also states: "With S5 and S12 plays an important role in translational accuracy."
uS4 is one of the primary binding proteins that initiates 30S ribosomal subunit assembly by binding directly to 16S rRNA. Based on articles retrieved from PubMed, Dutca et al. (2007) demonstrated that the complex of primary r-protein S4 and 16S rRNA undergoes a temperature-dependent conformational rearrangement, showing that S4 actively remodels the rRNA during early assembly: "a temperature-dependent conformational rearrangement was reported for a complex of primary r-protein S4 and 16S rRNA" [PMID:17376481, DOI:10.1016/j.jmb.2007.02.064]. These conformational changes in 16S rRNA are "consistent with subsequent downstream assembly events such as assembly of the secondary and tertiary binding r-proteins" PMID:17376481.
uS4 plays a central role in translational accuracy through its interaction with uS5 at the S4-S5 interface. According to PubMed, Agarwal et al. (2015) showed: "Ribosomal proteins S4 and S5 participate in the decoding and assembly processes on the ribosome and the interaction with specific antibiotic inhibitors of translation. Many of the characterized mutations affecting these proteins decrease the accuracy of translation, leading to a ribosomal-ambiguity phenotype" [PMID:25548247, DOI:10.1128/JB.02485-14]. The same study reported that "the tRNA selection pathway involves a transition between the closed and open conformations of the 30S ribosomal subunit and requires disruption of the interface between the S4 and S5 proteins" PMID:25548247.
Kamath et al. (2017) further characterized the C-terminus of uS4: "uS4 helps initiate the assembly of the small subunit by binding directly to 16S rRNA and at least some of the uS4 alterations that promote miscoding also display assembly defects" and "uS4 is also a translational repressor and is involved in transcription antitermination activities" [PMID:28483689, DOI:10.1016/j.biochi.2017.05.004]. This paper also showed that "the C-terminal requirements for ribosome assembly are less rigid than for mRNA decoding" PMID:28483689.
The P. aeruginosa uS4 protein (206 aa) has been resolved in multiple cryo-EM structures of the P. aeruginosa 70S ribosome:
PDB 7UNR/7UNU/7UNV/7UNW: 70S ribosome initiation complex bound to IF2-GDP/GDPCP [PMID:35697706, DOI:10.1038/s41467-022-31129-2]. These structures from Basu et al. (2022) show the complete P. aeruginosa 70S ribosome including uS4 at 2.6-2.9 A resolution.
PDB 8RWG: Additional P. aeruginosa ribosome structure at 2.46 A resolution.
Halfon et al. (2019) solved the structure of P. aeruginosa ribosomes from a clinical isolate, including the 30S subunit (PDB 6SPE) [PMID:31611393, DOI:10.1073/pnas.1909831116].
The protein contains:
- N-terminal domain (IPR001912, residues 3-95): uS4 N-terminal domain
- Conserved site (IPR018079, residues 94-118): uS4 conserved site at the junction between N-terminal and RNA-binding domains
- S4 RNA-binding domain (IPR002942, residues 96-160): canonical S4 fold for RNA recognition
- Bacterial uS4 family (IPR005709): full-length bacterial S4 family signature
While not directly demonstrated in P. aeruginosa, E. coli S4 has additional established functions:
- Translational autoregulation: S4 represses translation of its own mRNA and the alpha operon mRNA PMID:28483689
- Transcription antitermination: S4 has been implicated in NusA-dependent transcription antitermination PMID:28483689
Related studies in P. aeruginosa have characterized 30S assembly factors that work alongside uS4:
- RsgA (Small Ribosomal Subunit Biogenesis GTPase A) is involved in late stages of 30S maturation in P. aeruginosa [PMID:31199072, DOI:10.1111/febs.14959]
- RbfA (Ribosome-binding factor A) is a small ribosome assembly factor involved in 30S maturation [PMID:33157160, DOI:10.1016/j.bbagen.2020.129780]