almost like this! new ligase-derived rna polymerase ribozymes m. s. lawrence and d. p. bartel rna...
TRANSCRIPT
Almost like this!
New ligase-derived RNA polymeraseribozymes
M. S. Lawrence and D. P. BartelRNA (2005), 11:1173-1180
Almost like this!
An ’RNA World’
Univ. of Oregon, www.uoregon.edu/...
An ’RNA World’
(Wikimedia Commons)
”Primordial Sea”
An ’RNA World’
”Primordial Sea”Red Sea
”[…] the alternative scenario in which oligonucleotides and code protein synthesis emerged together in miraculous concert.” (p.1173)
Historical background
• 1967: Woese suggests that primordial organisms may have been based on RNA
• 1982: Cech et al. finds a self-splicing intron (a ribozyme)
• 1986: Gilbert introduces the term ’RNA World Hypothesis’
• 1989-92: Szostak/Doudna develops derivatives of self-splicing introns, but with limited succes
• 1992 og forth: Noller et al. demonstrates that rRNA is responsible for the peptidyl transferase activity
• 1993: Szostak/Ekland develops RNA-based ligases
• 2001: Bartel, Lawrence and others developribozymatic polymerases Pol 1 and Evolved Pol 1
Szostaks/Ekland (1993):
Design of the initial library
- the Class I RNA ligase is almost asfast as an enzyme (kcat ≈ 1 s-1),
- and it will do several turnovers
- but it requires that the template is base-paired onto it, i.e. it is not generic
Szostaks/Ekland (1993):
Design of the initial library
Lawrence/Bartel (2001):Extending the ligase domain with an auxiliary domain
Selection method
Goal: Select polymerases that incorporate rNTPs onto their ”template end”
Method:1. Tagged UTP: 4-thio-UTP2. Retard molecules with 4-thio-UTP on ”mercury gels” (acrylamide gel w/urea+N-acryloyl-aminophenylmercuric acetate)3. Excise and amplify
[Science 2001, 292:1319]
4-thio-UTP
Selection method
Goal: Select polymerases that incorporate rNTPs onto their ”template end”
Method:1. Tagged UTP: 4-thio-UTP2. Retard molecules with 4-thio-UTP on ”mercury gels” (acrylamide gel w/urea+N-acryloyl-aminophenylmercuric acetate)3. Excise and amplify
[Science 2001, 292:1319]
Selection method
• Elongation w/tagged rNTPs (vars: t, [rNTP], tag, turnovers)• Purification (APM-PAGE 1-2× or using biotin/streptavidin)• RT-PCR (error prone)• In vitro transcription of cDNA
Cloning and sequencing
Goal: Select polymerases that incorporate rNTPs onto their ”template end”
Method:1. Tagged UTP: 4-thio-UTP2. Retard molecules with 4-thio-UTP on ”mercury gels” (acrylamide gel w/urea+N-acryloyl-aminophenylmercuric acetate)3. Excise and amplify
[Science 2001, 292:1319]
Original selection (2003)
After 8 rounds, a number of distinct types (”families”) begin to emerge:
Fig. 2
Original selection (2003)
After 10 rounds, members of the Pol 1 family dominate:
Fig. 2
Original selection (2003)
Pol 1 optimised to polymerise at up to 14 nt, i.e. beyond one hel. turn!
Fig. 2
Revising the selection criteria
Lessons learned from that study:
1. While 4SUTP worked fine, biotin-ATP did not
2. Mercury gels allow for selection of incorporation of two 4SUs
3. 2-aminopurine in the template helps pairing with 4SU
Moreover:- competitors (A, C, G)- lower [4SUTP]- try various primer-templates
New selection (2005)
Fig. 2
Re-examining an earlier pool for alternative polymerases, now using the new selection criteria:
Pol 1 re-emerges, but is then overtaken!
New selection (2005)
Fig. 233 new families!
Re-examining an earlier pool for alternative polymerases, now using the new selection criteria:
Pol 1 re-emerges, but is then overtaken!
Pol 2 is the new Pol 1!
Examining the new polymerases
Polymerases are named according to their efficacy
Fig. 3
Fig. 4
Primer only
+ Ligase core (no aux.)
Examining the new polymerases
Polymerases are named according to their efficacy
Strong stem structures appear to be a requirement; computational folding finds that these folds are among the 70% most stable within each sequence space.
Conclusion: The ligase core can be extended with an auxiliary domain and become generic!
Fig. 3
Final notes
”Parasites” are a problem during selection:
Final notes
”Parasites” are a problem during selection
As much as 6 nt extension was achieved with non-optimised RNA polymerases. Authors suggest further experiments:
1. Optimisation (cf. Evolved Pol 1)2. A selection ”battle” between select polymerases3. Recombination during selection
Final notes
”Parasites” are a problem during selection
As much as 6 nt extension was achieved with non-optimised RNA polymerases
RNA polymerases are limited by their own degradation:
”The polymerization reaction uses micromolar concentrations of RNA, but [Pol 1] binds its PT substrate with only millimolar affinity” (p. 1178)
”As the ribozyme slowly polymerizes NTPs, it suffers the constant hydrolytic onslaught of the [200 mM Mg2+, pH 8.5] reaction buffer” (p.1178)
(Data not shown.)
Final notes
”Parasites” are a problem during selection
As much as 6 nt extension was achieved with non-optimised RNA polymerases
RNA polymerases are limited by their own degradation
- Bartel’s current work is focused on improving substrate interactions using micelles.
(Wikimedia Commons)
RNase A means ”The End”
Final notes
”Parasites” are a problem during selection
As much as 6 nt extension was achieved with non-optimised RNA polymerases
RNA polymerases are limited by their own degradation
- Bartel’s current work is focused on improving substrate interactions using micelles.
RNase A = ”The End”