Known Stalling Peptides

Validation against characterized prokaryotic ribosome stalling sequences

Overview

Several prokaryotic ribosome stalling peptides have been experimentally characterized. These serve as positive controls for our discovery pipeline.

SecM (Secretion Monitor)

Sequence

FSTPVWISQAQGIRAGP
       ↑      ↑↑↑
       W      Stall site

Characteristics

Property	Value
Organism	Escherichia coli
Length	17 residues
Stall motif	GIRAGP
Function	Secretion regulation
PDB	Multiple structures

Mechanism

SecM stalls ribosomes when the SecY translocon is unavailable, allowing SecA upregulation.

flowchart LR
    mRNA[secM-secA mRNA] --> Ribosome
    Ribosome --> |SecY available| Continue[Translation continues]
    Ribosome --> |SecY blocked| Stall[Ribosome stalls]
    Stall --> |Structure change| Expose[secA RBS exposed]
    Expose --> SecA[SecA synthesis]

Figure 1: SecM regulatory mechanism

TnaC (Tryptophanase Leader)

Sequence

WDPXXXXIGP (consensus)
↑         ↑↑
W         Stall (tryptophan-dependent)

Characteristics

Property	Value
Organism	Escherichia coli
Length	Variable (~24 residues)
Stall motif	IGP (tryptophan-dependent)
Function	Tryptophan catabolism
Inducer	L-tryptophan

Mechanism

TnaC stalling requires free L-tryptophan in the ribosome exit tunnel, linking amino acid availability to gene expression.

MifM (Membrane Insertion Factor Monitor)

Sequence

XXXGPXXGIAGP
   ↑↑    ↑↑↑↑
   GP    Stall site

Characteristics

Property	Value
Organism	Bacillus subtilis
Length	~90 residues (full peptide)
Stall motif	GIAGP
Function	YidC regulation
Homologs	Many Firmicutes

Mechanism

MifM monitors membrane protein insertion via YidC. When insertion is impaired, stalling upregulates YidC2.

CydA (Cytochrome bd Oxidase)

Sequence

XXXRAGP
   ↑↑↑↑
   Stall site

Characteristics

Property	Value
Organism	Escherichia coli
Length	Short leader
Stall motif	RAGP
Function	Cytochrome regulation

Comparison Table

Peptide	Motif	Trigger	Response
SecM	GIRAGP	SecY absence	SecA↑
TnaC	IGP	Trp presence	TnaAB↑
MifM	GIAGP	YidC absence	YidC2↑
CydA	RAGP	Unknown	CydB?

Validation Strategy

Positive Control

Our HMMs should recover known stalling peptides:

1. Search prokaryotic databases with seed HMMs
2. Check for known peptides in results
3. Measure E-values and ranks

Expected Results

Peptide	Expected E-value	Status
SecM	<1e-10	TBD
TnaC	<1e-5	TBD
MifM	<1e-8	TBD
CydA	<1e-5	TBD

Negative Control

Random GP sequences should not rank highly:

• Shuffle upstream context
• Preserve amino acid composition
• Compare E-value distributions

Novel Discoveries

Criteria for Novel Peptides

1. Sequence: Contains xAGP or similar motif
2. Context: Inter-domain location
3. Conservation: Conserved across species
4. Structure: Predicted stalling-compatible

Candidate Validation

Promising candidates should be validated by:

1. Ribosome profiling correlation
2. Reporter assays (if available)
3. Structural modeling
4. Phylogenetic analysis

References

1. Ito K, Chiba S. (2013) Arrest peptides: cis-acting modulators of translation. Annu Rev Biochem 82:171-202.
2. Wilson DN, et al. (2016) Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat Rev Microbiol 14:22-35.
3. Seip B, Innis CA. (2016) How Widespread is Metabolite Sensing by Ribosome-Arresting Nascent Peptides? J Mol Biol 428:2217-2227.