Prolonged translational arrest in Escherichia coli results in cleavage of the codon present in the A-site of paused ribosomes. A-site mRNA cleavage facilitates tmRNA·SmpB recruitment and subsequent recycling of the stalled ribosome complex. In this dissertation, I demonstrate that the aminoglycoside antibiotics inhibit A-site mRNA cleavage of stop codons during inefficient translation termination. Consistent with their known miscoding properties, aminoglycosides increased stop codon readthrough, which was accompanied by a decrease in tmRNA·SmpB activity. A-site cleavage in streptomycin-resistant and streptomycin pseudodependent cells treated with streptomycin was indistinguishable from wild-type cells. However, streptomycin dependent cells exhibited significantly lower levels of A-site mRNA cleavage. Suppressor mutations in rpsD, encoding ribosomal protein S4, restored A-site mRNA cleavage to streptomycin dependent cells. Taken together, these results demonstrate that A-site dynamics significantly influence A-site mRNA and tmRNA·SmpB activity.
The results presented in the rest of this dissertation suggest that Gram-positive bacteria have a distinct family of RNAse and immunity proteins that are related to contact-dependent inhibition (CDI) in Gram-negatives. CDI in proteobacteria is mediated by stick-like proteins on the cell surface which deliver toxic tips into target cells. The protein family associated with CDI consists of proteins CdiA, CdiB, and CdiI. We discovered that CdiA protein EC869 and YwqJA from the Gram-positive organism Bacillus pumilus share related C-terminal sequences which suggests that these proteins may be functionally analogous. YwqJA is a member of a novel family of conserved proteins (UPF0720) with similar amino-terminal domains and highly variable carboxy-termini. UPF0720 proteins inhibit colony formation when immunity protein is specifically degraded. Purified UPF0720 proteins have RNAse activity in cell-free extracts and this RNAse activity is blocked by immunity. During starvation, the UPF0720 protein YobL is extracellular where it may interact with target cells. Based on evidence shown here, we have extended our knowledge of CDI to Gram-positive organisms and identified a group of RNAses that possess many characteristics of CDI-associated proteins.
|Commitee:||Low, David, Perona, John, Wilson, Leslie|
|School:||University of California, Santa Barbara|
|Department:||Molecular, Cellular & Developmental Biology|
|School Location:||United States -- California|
|Source:||DAI-B 73/03, Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology|
|Keywords:||Antibacterial chemotherapeutics, Antibiotics, Natural toxins, Ribonuclease, Ribosome, Translation|
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