Single Molecule Förster Resonance Energy Transfer and Super Resolution Force Spectroscopy Study of Elongation Factor G Evolution, Conformation, and Function

The ribosome is a ribonucleoprotein complex consisting of two subunits. During protein synthesis the ribosome decodes mRNA facilitated by a translocase enzyme, elongation factor G (EF-G). EF-G induces the advancement of the mRNA reading frame by 3 nucleotides (nt) and tRNA translocation via the hydrolysis of one GTP molecule. The mechanism of this reaction remains largely unknown. Cryo-EM studies indicated that the switch I region of EF-G undergoes major conformational changes following GTP hydrolysis, which may drive tRNA translocation. Multiple sequence alignment (MSA) of switch I identified a highly conserved threonine (Thr48) in prokaryotes, which may be homologous with Thr56 of eukaryotes. Two mutations were prepared: a phosphomimic (T48E) and a nonpolar substitution (T48V). Biological assays indicated both inhibited tRNA translocation. Single molecule Förster resonance energy transfer (smFRET) experiments determined this inhibition stems from the mutants becoming trapped in a ‘super’-compact conformation on the ribosome. The Thr48 mutations were modeled using AlphaFold predictions and MSAs. This revealed the mutations shifted the conformational balance to two previously reported EF-G conformations, con1 and con2. Predicted ancestral analogues were generated using phylogenetic tree construction. Two analogues resembling con1 and con2 were expressed. Both analogues exhibited minimal GTPase activity. Translocation fidelity was measured using super resolution force spectroscopy (SURFS), which found that both maintained the proper 3-nt reading frame. Structural studies have shown Q508 and H584 in domain IV of EF-G play important roles in translocation as they interact with codon-anticodon minihelix formed between mRNA and the A-site tRNA. We investigated EF-G mediated power stroke and translocation fidelity via mutagenesis of these two residues. Five substitution mutations were expressed (H584K, H584E, H584Q, Q508K, and Q508E). Biological assays have shown that all mutants retain uncompromised GTPase activity. Translocase activity was measured using SURFS. While Q508K maintained the proper 3-nt reading frame and produced normal power-stroke force, H584K induced a ‘-1’-nt frameshift and adversely affected power-stroke generation. smFRET experiments also indicated both mutations inhibit EF-G’s ability to enter a compact conformation when not bound to the ribosomal complex. Taken together, these studies provide us with new knowledge of GTPase coupled translocation and power-stroke force.

Name thesis:degree_name

Doctor of Philosophy

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Houston

Year dc:date.issued

2025

Author dc:creator

• Steele, Jacob Hunter 1993-

Advisor dc:contributor.advisor

• Wang, Yuhong

Committee members dc:contributor.committeemember

• Briggs, James M.
• Widger, William R.
• Morrison, Greg

• Biochemistry
• Biology

Language dc:language.iso

English

Handle dc:identifier.uri

https://hdl.handle.net/10657/20590

OAI identifier oai:identifier

oai:uh-ir.tdl.org:10657/20590