Date of Award
2024-08-01
Degree Name
Master of Science
Department
Chemistry
Advisor(s)
Eda Koculi
Abstract
The ribosome is the multimegadaltons RNA-protein complex responsible for protein production in every known organism. Propper ribosome assembly is required for protein production to occur efficiently and accurately. RNA provides the platform for the ribosome assembly. Dozens of protein maturations factors facilitate RNA folding, post-transcriptional modifications and processing during ribosome assembly in bacterial cells. A complete understanding of ribosome assembly in cell requires the systematic characterization of all the RNA folding, modifications, maturation, processing events, and how are all these RNA maturation events are synchronized both in time and space.In cells, bacterial ribosome assembly occurs quickly, with short-lived ribosomal intermediates. To increase the accumulation of ribosomal intermediates in cells for biochemical and structural investigations, the helicase inactive R331A DbpA construct was implemented. DbpA is an Escherichia coli (E. coli) DEAD-box RNA helicase involved in large subunit ribosome assembly in bacteria. When R331A DbpA is expressed in cells, three large subunit ribosomal intermediates with sedimentation coefficients of 27S, 35S and 45S accumulate. Using Oxford Nanopore Technologies Direct RNA Sequencing kit, the RNA modifications and processing of 35S and 45S intermediates present in the E. coli cells expressing R331A DbpA were investigated. Furthermore, using multiple gene deletion and different growth conditions, the role of Hfq and ProQ proteins in ribosome assembly and how their functions are interconnected to the DbpA proteinâ??s function during the ribosome maturation process was investigated.
Language
en
Provenance
Received from ProQuest
Copyright Date
2024-08-01
File Size
80 p.
File Format
application/pdf
Rights Holder
Isaac Samuel Weislow
Recommended Citation
Weislow, Isaac Samuel, "Investigation of Ribosome Assembly in Escherichia Coli via Oxford Nanopore Technologies Direct RNA Sequencing and Targeted Gene Modification" (2024). Open Access Theses & Dissertations. 4214.
https://scholarworks.utep.edu/open_etd/4214