Functional Characterization of Ribosome Maturation Factor RimP in Mycobacterium smegmatis


Student thesis: Doctoral Thesis

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  • Xing WENG

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Award date16 Jun 2016


Ribosome assembly is a very complicated process in bacteria, including folding of ribosomal RNA (rRNA) and incorporation of ribosomal proteins onto the rRNA. A large number of assembly and maturation factors are required for the efficiency and accuracy of ribosome assembly. Ribosome maturation factor RimP is one of the maturation factors that involved in the maturation of 30S subunit in Escherichia coli (E.coli). Despite the importance of RimP in bacteria, the functional study of RimP in mycobacteria is limited. Mycobacterium tuberculosis (Mtb) is the pathogen that causes the notorious respiratory infectious disease, Tuberculosis (TB). Mycobacterium smegmatis (Msmeg), as a model for studying Mtb, is widely used due to its fast growth and safe to culture in the lab, so Msmeg is used in our study. RimP is conserved among different species of mycobacteria. RimP in Msmeg and Mtb share 61% amino acid sequence identity, possess identical secondary structures between each other. This implies that the function of RimP between Msmeg and Mtb are similar, if not the same. Structural prediction of RimP shows a Sm-like C-terminal domain that can interact with RNA. In this thesis, functional characterization of RimP was carried out from four aspects: determination of RNA binding ability, identification of protein binding partners, functional characterization on ribosome maturation and stress resistance.
The structure of RimP from Msmeg was determined previously using X-ray crystallography, which consists of N-terminal domain and C-terminal domain. The N-terminal domain possesses two α-helices and three β-sheets, and the C-terminal domain contained two α-helices and five β-sheets. The N-terminal domain is a ribosomal domain whereas the C-terminal domain possesses an Sm-like structure. RNA binding assay showed that no RNA bound to RimP in vitro. Therefore, we focus on protein-binding function of N-terminal domain in the next step.
As protein binding partners could help to explore the functional roles of target protein, protein binding partners of RimP with potential biological significance were identified. By co-immunoprecipitation followed by mass spectrometry, numerous candidates bound to RimP were found. Using in vitro co-expression and pull down assay, we identified that ribosomal protein RpsL (S12) directly bound to RimP with specificity and high affinity.
70S ribosome is consist of 30S small subunit and 50S large subunit. Ribosomal protein RpsL (S12) is component of 30S small subunit. In order to map the specific interaction sites on the two proteins, multiple truncated forms of each protein were constructed to test the binding affinity. According to the mapping result, N-terminal truncated form of RpsL bound to full-length RimP, which indicated that the truncated N-terminal part was not necessary for binding; On the other hand, N-terminal or C-erminal domain alone of RimP could not bind to full-length RpsL, which implied that neither N-terminal nor C-terminal domain of RimP could independently bind to RpsL. As the linker that connected N-terminal and C-terminal domains could be involved in protein-protein interaction, 11 mutations of linker were constructed. Based on the binding assay, flexibility or charge changes of two specific amino acids on the linker could alter the binding affinity with RpsL, suggesting their roles in facilitating the binding of RpsL by either alternating the orientation of N-terminal and C-terminal domains or involving in the interaction with residues in RpsL. Interestingly, compared with other amino acids that not directly involved in protein binding affinity, the two amino acids showed higher conservation among gram-positive and gram-negative bacteria, suggesting the importance of the two amino acids in RimP-RpsL binding and in turn in ribosome biogenesis.
In the process of ribosome assembly, RpsL is incorporated into 30S subunit by interaction with 16S rRNA and other ribosomal proteins. To investigate how RimP affects RpsL-16S rRNA interaction, in vitro ribosomal RNA binding was tested with RimP, RpsL or RimP-RpsL complex respectively. It was shown that the binding affinity of RpsL to both 23S rRNA and 16S rRNA was weak. RimP could bind to neither 23S rRNA nor 16S rRNA. However, RimP-RpsL complex bound to the rRNAs with a largely increased affinity. Therefore, we conclude that RimP could assist assembly of RpsL into 30S subunit by improving RpsL affinity to 16S rRNA.
To investigate the effect of RimP on the ribosome maturation in bacteria, polysome profiling was performed. The distribution of ribosome subunits, ribosomes and polysomes in the polysome profile could represent the in vivo situation of ribosome maturation. As reported, rimP knockout (ΔrimP) mutant of E.coli showed significant difference of peak height of 30S subunit, 50S subunit, 70S ribosome and polysomes compared to wild type. In Msmeg, ΔrimP mutant showed slightly higher level of 30S subunit, a remarkable increase in 50S subunit and decreased amount of 70S ribosome compared to wild type (WT), which revealed that the maturation of 70S ribosome was impaired in the absence of rimP. Additionally, the decreased level of polysomes indicated the defective translation efficiency in ΔrimP mutant.
Once infected by Mtb, the human body would release macrophage to attack Mtb by reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI). In this study, response of Msmeg to nitrosative and oxidative stresses induced by RNI and ROI were investigated in the presence and absence of RimP. In Msmeg, ΔrimP mutant showed lower viability in response to RNI stress than WT. Besides, WT showed higher survival rate than ΔrimP mutant in macrophage invasion assay, suggesting the significant role of RimP in the detoxification of RNI in Msmeg and survival of Msmeg in macrophage. With treatment of RNI, WT showed reduced peak area of 70S ribosome, however, ΔrimP mutant showed severely reduced peak area of 30S subunit, 50S subunit, 70S ribosome. Therefore, the reduction in RNI resistance was associated with ΔrimP-induced defects in ribosome maturation.
In summary, our results reveal the significance of RimP in ribosome maturation and RNI resistance, which have important implications for an understanding of RimP functions in Mtb. This information provides foundation for the future development of therapeutic approaches using RimP as a druggable target against mycobacterial infection.