DEAD-box helicases form nucleotide-dependent, long-lived complexes with RNA.

Area of Science:

• Molecular biology
• Biochemistry
• RNA biology

Background:

• DEAD-box RNA helicases are crucial enzymes that remodel RNA and RNA-protein complexes.
• These helicases are known to form stable complexes with RNA, a process termed RNA clamping.
• The underlying molecular mechanisms of RNA clamping remain incompletely understood.

Purpose of the Study:

• To investigate the molecular basis of RNA clamping by DEAD-box RNA helicases.
• To characterize the formation and stability of complexes between yeast DEAD-box helicases and RNA.
• To explore the role of ATP binding states in RNA clamping.

Main Methods:

• Utilizing non-hydrolyzable ATP analogues (ADP-BeFx and ADP-AlFx) to stabilize specific nucleotide-binding states.
• Employing biochemical assays to measure the lifetimes of helicase-RNA complexes.
• Introducing specific mutations (e.g., R489A) in DEAD-box helicases to probe their function.

Main Results:

• The yeast DEAD-box helicase Ded1p forms exceptionally long-lived complexes with RNA in the presence of ADP-BeFx or ADP-AlFx, lasting several hours.
• Neither the nucleotide nor Mg(2+) is released from these stable complexes.
• A mutation in Ded1p (R489A) disrupted long-lived complex formation with the ATP transition state analogue but not the ground state analogue.
• Other DEAD-box helicases (Mss116p, Sub2p) also formed long-lived complexes with ADP-BeFx, but differential binding to ADP-AlFx was observed.
• All tested DEAD-box helicases exhibited persistent RNA binding in the ATP ground state.

Conclusions:

• DEAD-box RNA helicases possess an inherent capacity to function as potent, ATP-dependent RNA clamps.
• RNA clamping is a conserved feature, particularly evident in the ATP ground state across different DEAD-box helicases.
• The ATP transition state may play varied roles in RNA binding dynamics for distinct DEAD-box helicases.