1. Stringent Response in Streptomyces coelicolor
Differentiation: formation of aerial mycelia, followed by sporulation
Antibiotics also produced near time of differentiation

2. Streptomyces rel mutants
S. equisilimus RelA - properties of both E. coli’s SpoT and RelA
S. coelicolor homolog (formerly ORF1)
discovered when deletion prevented antibiotic production
strong homology to E. coli proteins

3. Possible Homology Situations
Concentrated at N-terminus
Concentrated at C-terminus
Consistent throughout

4. E. coli RelA and E. coli SpoT
E. coli RelA and S. coelicolor Rel
Identities = 278/734 (37%), Positives = 425/734 (57%), Gaps = 53/734 (7%)
E. coli SpoT and S. coelicolor Rel
Identities = 295/741 (39%), Positives = 429/741 (57%), Gaps = 49/741 (6%)
E. coli RelA and E. coli SpoT
Identities = 216/680 (31%), Positives = 354/680 (51%), Gaps = 39/680 (5%)

5. Correlation between structure and function?
Not apparent from sequence homologies
Expressed in E. coli, has SpoT properties
In S. coelicolor mutants, has both RelA and SpoT properties (similar to S. equisilimus relA)
Deletion mutants may elucidate a structure/function connection, if one exists

6. Deletion mutants spoT rel relA rel Full length Δ(1-92) Δ(1-229)
Δ(ATP/GTP)
Δ( )
Δ(1-358, )
Δ(1-229, )
Δ(1-266, )

7. Chromatography basics
F1blue – F2blue = ΔFblue
Chromatography basics
F1red – F2red = ΔFred
ΔFblue
ΔFred
F1 : Impelling force
hydrodynamic
Polar and steric interactions between the solvent and solute determine the degree of separation.
F2: Repelling forces
Surface energy, Van der Waals forces, steric specificity

8. ppGpp production Positive result indicates ppGpp was detected.
Full length
Δ(1-92)
Δ(1-229)
Δ(ATP/GTP)
Δ( )
Δ(1-358, )
Δ(1-229, )
Poor expression
Δ(1-266, )
Positive result indicates ppGpp was detected.

9. ppGpp accumulation
Full length
Δ(1-92)
Δ(1-229)
Δ(ATP/GTP)
Δ( )
Δ(1-358, )
n/a
Δ(1-229, )
n/a
Poor expression
Δ(1-266, )
Positive result indicates ppGpp content increased after addition of serine hydroxamate, indicating dependence on ribosomes.

10. possible ribosome binding modulatory region
ATP/GTP binding motif
catalytic domains
NH2
COOH 1 93
267
397
453
847
possible ribosome binding modulatory region
phosphohydrolase
synthetase