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Bacteriophage lambda N gene leader RNA. RNA processing and translational initiation signals
D A Steege1, K C Cone, C Queen
1Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
The Journal of Biological Chemistry
|December 25, 1987
Summary
Researchers mapped RNA processing sites in bacteriophage lambda's PL transcript. They identified RNase III cleavage sites and two ribosome binding sites, shedding light on N protein expression regulation.
Area of Science:
- Molecular Biology
- Virology
- RNA Processing
Background:
- Bacteriophage lambda gene expression is regulated by complex RNA structures and processing events.
- RNase III is a key enzyme involved in RNA processing, often recognizing double-stranded RNA structures.
- Ribosome binding sites (RBS) are crucial for translational initiation and regulation.
Purpose of the Study:
- To precisely map RNase III processing sites and ribosome binding sites within the bacteriophage lambda major leftward promoter (PL) transcript.
- To elucidate the secondary structure of the RNase III recognition site and its implications for cleavage.
- To investigate the role of identified ribosome binding sites in translational regulation of N gene expression.
Main Methods:
- In vitro transcription of the bacteriophage lambda PL transcript.
- Enzymatic cleavage using purified RNase III.
- Structure mapping with single- and double-stranded RNA-specific nucleases.
- In vitro ribosome binding assays.
Main Results:
- Two specific endonucleolytic cleavages by RNase III were mapped at nucleotides 88 and 197 of the PL transcript.
- RNA structure mapping provided evidence for a stem-loop structure at the N recognition (nutL) site, consistent with RNase III processing.
- Efficient in vitro ribosome binding was observed at the region containing the AUG codon at position 223, supporting its role as the N protein start codon.
- An additional ribosome binding site was identified downstream of the nutL site, suggesting a potential second site for translational control.
Conclusions:
- The study precisely defines RNase III processing sites and secondary structures within the bacteriophage lambda PL transcript.
- The findings confirm the AUG at position 223 as the N protein start codon and reveal a second potential ribosome binding site.
- These identified sites and structures offer insights into the intricate regulation of phage development and N gene expression through RNA processing and translational control.