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Related Experiment Videos

Dual translational initiation sites control function of the lambda S gene.

U Bläsi1, K Nam, D Hartz

  • 1Department of Biochemistry and Biophysics, Texas A and M University, College Station 77843.

The EMBO Journal
|November 1, 1989
PubMed
Summary

This study explores how gene S in phage lambda controls lysis timing through dual translational initiation. Using in vitro methods, the researchers found that two stem-loop structures regulate initiation at two start codons, Met1 and Met3. They discovered that Shine-Dalgarno sequences direct ribosome binding to each start codon. The ratio of initiation events correlates with lysis timing in vivo. Mutant alleles showed altered lysis timing that matched in vitro findings. The study suggests that two polypeptides with opposing functions regulate lysis. This mechanism is unique among phage genes and highlights the role of RNA structure in translational control.

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Area of Science:

  • Molecular genetics
  • Gene expression regulation
  • Bacteriophage biology

Background:

The regulation of gene expression in bacteriophages is a well-studied field, yet specific mechanisms governing translational control remain unclear in some cases. Prior research has shown that phage lambda genes often rely on complex regulatory elements to coordinate lysis and lysogeny. However, the precise role of dual translational initiation sites in gene S has not been fully resolved. It was already known that gene S contributes to lysis timing, but how initiation site partitioning affects this process was uncertain. This gap motivated further investigation into the structural elements controlling translation of gene S. No prior work had resolved how Shine-Dalgarno sequences might interact with multiple start codons. This uncertainty left open the possibility that structural RNA elements could influence initiation site selection. Understanding this mechanism could clarify how phage lambda balances lysis and lysogeny at the translational level.

Purpose Of The Study:

This study aimed to investigate the regulation of gene S in phage lambda by focusing on translational initiation sites. The specific problem addressed was how two adjacent start codons contribute to lysis timing. The motivation arose from genetic data suggesting dual initiation events. The goal was to determine if structural RNA elements control partitioning between the two start codons. The researchers proposed that Shine-Dalgarno sequences and stem-loop structures might regulate initiation site selection. They sought to test this hypothesis using in vitro methods. The study focused on correlating structural features with translational outcomes. The ultimate aim was to understand how gene S achieves functional diversity through dual initiation.

Keywords:
gene regulationphage lambdatranslational initiationShine-Dalgarno sequences

Frequently Asked Questions

Two stem-loop structures regulate initiation site selection in gene S. One is upstream of the gene, and the other is about 10 codons downstream.

Two adjacent Shine-Dalgarno sequences direct ribosome binding to Met1 and Met3. This allows initiation at both start codons.

The second stem-loop is located approximately 10 codons into the gene. It helps control the partitioning of translational initiations.

The ratio of ternary complex formation at Met1 and Met3 correlates with lysis timing in vivo. This supports the model of initiation site partitioning.

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Main Methods:

The researchers used in vitro techniques to examine translational initiation in gene S. They employed primer-extension inhibition, also known as 'toeprinting', to detect ribosome pausing. Two stem-loop structures were identified as potential regulators of initiation site selection. One stem-loop was located upstream of the reading frame, while the second was positioned approximately 10 codons into the gene. The team analyzed the Shine-Dalgarno sequences associated with each start codon. They tested the hypothesis that these sequences direct ribosomes to either Met1 or Met3. The study also measured ternary complex formation at both initiation sites. Correlations between in vitro translation and in vivo lysis timing were assessed using mutant alleles.

Main Results:

The study revealed that two stem-loop structures regulate translational initiation in gene S. One stem-loop is positioned immediately upstream of the gene, while the second is located about 10 codons downstream. Using toeprinting, the researchers confirmed that two adjacent Shine-Dalgarno sequences serve the start codons Met1 and Met3. Translational initiation occurs at both sites, generating S107 and S105 polypeptides. The ratio of initiation events at Met1 and Met3 correlates with lysis timing in vivo. Mutant alleles showed altered lysis timing that matched in vitro ternary complex formation ratios. The findings suggest that initiation site partitioning controls lysis scheduling. Structural RNA elements appear to direct ribosome binding to specific start codons.

Conclusions:

The authors propose that the regulation of gene S depends on the partitioning of translational initiations between two start codons. Structural RNA elements upstream and within the gene control this partitioning. Shine-Dalgarno sequences adjacent to Met1 and Met3 direct ribosome binding. The ratio of initiation events influences lysis timing in vivo. The study supports a model where two polypeptides with opposing functions regulate lysis. The findings suggest that dual initiation allows for fine-tuned control of gene S activity. The researchers conclude that this mechanism is unique among phage genes. The study highlights the importance of RNA structure in translational regulation.

Mutant alleles alter lysis timing in vivo. These changes match in vitro ternary complex formation ratios, supporting the model.

The authors propose that gene S is unique in that two polypeptides with opposing functions regulate lysis. This is not observed in other phage genes.