Difference between revisions of "Part:BBa K567001"

Latest revision as of 00:49, 31 October 2011

lacI-Ptrc-tRNA(Arg)

The tRNA^Arg is under the control of promoter trc. tRNA^Arg expression is induced by 0.5mM IPTG when the OD600 of the culture reaches 0.3.

This part is used to control the translation process by over expressing tRNA^Arg, and to cooperate with related parts to analyse and characterize two factors in rare-codon switch system.

Control Rare tRNA amount

Design

In this part we have overexpressed rare tRNA^Arg-AGG in the cell. The rare tRNA can recognize AGG codon on the mRNA.

tRNA^Arg-AGG(BBa_K567001): tRNA^Arg-AGG is over expressed under the control of trc promoter (induced by IPTG).

This rare tRNA^Arg can be charged with Arg by native Arginyl-tRNA Synthetase(ArgRS) in E.coli.

Over expressed tRNA(Arg)-AGG is charged by native Arginyl-tRNA Synthetase(ArgRS)

RFP-6AGG(BBa_K567017): we have inserted 6 AGG codons after the start codon ATG in the RFP gene.

6AGG codons are inserted after the start codon ATG in the reporter gene

Action

When rare tRNA^Arg-AGG is not over-expressed, RFP expression is hindered. When tRNA^Arg-AGG is over-expressed, this tRNA can recognize the AGG codon on the mRNA so a large amount of RFP is produced.

Result

Fig.1 Confocal Microscope examining RFP expression. RFP has been largely produced in cells overexpressing tRNA^Arg-AGG.

Fig.2 Cells over-expressing tRNA^Arg-AGG emit bright red fluorescence as wild type RFP (first one from the left). Control (first one from the right) exhibits no red fluorescence.

RFP has been largely produced in cells overexpressing tRNA^Arg. No RFP can be observed in cells without rare tRNA overexpression.

Three Factor in Rare-Codon Switch

Number of Rare Codons

In this part we want to explore the influence of the number of rare codons inserted in the mRNA. We have inserted 2, 4, 6, 8 AGG codons respectively after the start codon in luciferase gene. T7 promoter or bla promoter^[1] are used to control target protein mRNA amount. We use different combinations of number of AGG codons and strength of promoters to characterize regulation^[1].

1) bla promoter-luciferase (weaker promoter)

A tandem of 2, 4, 6 or 8 AGG codons is inserted after the ATG codon of wild type luciferase

Parts:

Pbla-Luc-2AGG(BBa_K567004)

Pbla-Luc-4AGG(BBa_K567005)

Pbla-Luc-6AGG(BBa_K567006)

Pbla-Luc-8AGG(BBa_K567007)

2) T7 promoter-luciferase (stronger promoter)

A tandem of 2, 4, 6 or 8 AGG codons is inserted after the ATG codon of wild type luciferase

Parts:

PT7-Luc-2AGG(BBa_K567008)

PT7-Luc-4AGG(BBa_K567009)

PT7-Luc-6AGG(BBa_K567019)

PT7-Luc-8AGG(BBa_K567010)

Influence of inserted AGG codon number

The influence of different number of rare codons in regulating protein biosynthesis is shown below:

Fig.3 (A) Comparing the influence of different number of rare codon insertions in luciferase production. Pbla-Luc-4AGG (BBa_K567005), Pbla-Luc-6AGG (BBa_K567006) and Pbla-Luc-8AGG (BBa_K567007). (B) Comparing the influence of different number of rare codon insertions in luciferase production. Four Reporters are examined, including PT7-Luc-2AGG(BBa_K567008), PT7-Luc-4AGG(BBa_K567009), PT7-Luc-8AGG (BBa_K567010) and PT7-Luc-6AGG (BBa_K567019).

This picture reflects more clearly that the more rare codons are inserted, the lower the background expression and the narrower the range of device regulation. We are able to predict the outcome of influence of different number of rare codons in protein biosynthesis, offering valuable information for device usage.

Fig.4 The comparison between background expression and induced expression of luciferase with different rare codon insertions. (A)PT7-luc reporters. (B) Pbla-luc reporters

Influence of different strengths of target protein promoters

We examined the influence of different Reporter promoters on the working curve of our device, which is reflected by luciferase activity. The working range of our device is pre-defined by the strength of target protein promoter, T7 promoter and bla promoter in our project.

Fig.5 (A) Luciferase (Pbla-Luc-8AGG) production in cells overexpressing rare tRNA with lacI-Ptrc-tRNA^Arg. Luciferase production is reflected by bioluminescence emitted from the luciferin reaction. (B) Luciferase production in cells with PT7-Luc-8AGG as reporter. Here we analyze the influences of strong/weak promoter in luciferase production. Strong promoter (T7) of target gene can improve the titration curve, indicating that our device works better under strong target protein promoters.

Location of Rare Codons

Experiment results showed that when there is 111bp interval between the two-copy 4AGG, background is lower than there is a 30bp interval. In another analysis, protein production can be induced to a higher level when there are two copies of AGG tandems.

Fig.6 A. When there is 111bp interval between the two-copy 4AGG, background is lower than there is a 30bp interval. B. Protein production can be induced to a higher level when there are two copies of AGG tandems.

Experiment results showed that luciferase tagged with two-copy 4AGG insertions with an interval of 111 bp can increase yield and lower background noise of target protein. We further conducted experiments to test its characters.

When we compare the two-copy 4AGG tagged luciferase with the single copy one, we find that two-copy 4AGG tagged luciferase has a higher yield.

fig.7 Two-copy 4AGG tagged luciferase has a higher yield than single-copy

When we compare the two-copy 4AGG tagged luciferase with 8AGG tagged luciferase, we find that two-copy 4AGG tagged luciferase has a lower background.

fig.8 Two-copy 4AGG tagged luciferase has a lower background than 8AGG tagged luciferase

Related Biobrick

Pbla-Luc-2AGG (BBa_K567004)

Pbla-Luc-4AGG (BBa_K567005)

Pbla-Luc-6AGG (BBa_K567006)

Pbla-Luc-8AGG (BBa_K567007)

PT7-Luc-2AGG (BBa_K567008)

PT7-Luc-4AGG (BBa_K567009)

PT7-Luc-6AGG (BBa_K567019)

PT7-Luc-8AGG (BBa_K567010)

PT7-RFP-6AGG (BBa_K567017)

PT7-Luc-2x4AGG(111bp) (BBa_K567021)

PT7-Luc-2x4AGG(30bp) (BBa_K567026)

PT7-Luc-3x4AGG(30bp+111bp) (BBa_K567027)

Reference

Ulrich Deuschlel., et al., Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures The EMBO Journal vol.5 no. 11 pp.2987-2994, 1986

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 1845
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 1845
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 1845
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 1845
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 2026

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K567001 short</partinfo>
-The tRNA(Arg) is under the control of promoter trc. tRNA(Arg) expression is induced by 0.5mM IPTG when the OD600 of the culture reaches 0.3.
+The tRNA<sup>Arg</sup> is under the control of promoter trc. tRNA<sup>Arg</sup> expression is induced by 0.5mM IPTG when the OD600 of the culture reaches 0.3.
-=='''Background'''==
+This part is used to control the translation process by over expressing tRNA<sup>Arg</sup>, and to cooperate with related parts to analyse and characterize two factors in rare-codon switch system.
-'''tRNA Modulator''' and '''Reporter''' for Quantitative Analysis work together to regulate protein biosynthesis.
-'''tRNA Modulator''' controls rare tRNA amount. In our project, we use ''sulA'' promoter ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567002 BBa_K567002] ''sulA'' promoter-tRNA<sup>Arg</sup>-AGG) or  trc  promoter([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]'' lacI''-Ptrc-tRNA<sup>Arg</sup>) to control rare tRNA amount.
+=='''Control Rare tRNA amount'''==
-'''tRNA Modulator''':
+===Design===
-*''sulA'' promoter-tRNA<sup>Arg</sup>-AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567002 BBa_K567002])
+In this part we have overexpressed rare tRNA<sup>Arg</sup>-AGG in the cell. The rare tRNA can recognize AGG codon on the mRNA.
-*''lacI''-Ptrc-tRNA<sup>Arg</sup>-AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001] )
+'''tRNA<sup>Arg</sup>-AGG'''([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]): tRNA<sup>Arg</sup>-AGG is over expressed under the control of trc promoter (induced by IPTG).
-'''Reporter''' can control two elements: the number of rare codons inserted into luciferase after start codon ATG and the strength of target protein promoter.
+This rare tRNA<sup>Arg</sup> can be charged with Arg by native '''Arginyl-tRNA Synthetase(ArgRS)''' in E.coli.
-In our project, 2, 4, 6, 8 rare codon AGGs are inserted into the Reporter gene. T7 promoter or bla promoter are used to control target protein mRNA amount.
-'''Reporter''':
+[[image:11SJTU_Rare_03.jpg|center|Over expressed tRNA(Arg)-AGG is charged by native Arginyl-tRNA Synthetase(ArgRS)]]
+'''RFP-6AGG'''([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567017 BBa_K567017]): we have inserted 6 AGG codons after the start codon ATG in the RFP gene.
+[[image:11SJTU_rare_00.jpg|500px|center| 6AGG codons are inserted after the start codon ATG in the reporter gene]]
+===Action===
+When rare tRNA<sup>Arg</sup>-AGG is not over-expressed, RFP expression is hindered. When tRNA<sup>Arg</sup>-AGG is over-expressed, this tRNA can recognize the AGG codon on the mRNA so a large amount of RFP is produced.
+[[image:11SJTU_rare_01.jpg|center]]
+-----
+===Result===
+[[Image:11SJTU_rare_02.png|thumb|500px|center|''Fig.1'' Confocal Microscope examining RFP expression. '''RFP has been largely produced in cells overexpressing tRNA<sup>Arg</sup>-AGG.''']]
+[[Image:11SJTU_rare_10.jpg|thumb|500px|center|''Fig.2'' Cells over-expressing tRNA<sup>Arg</sup>-AGG emit bright red fluorescence as wild type RFP (first one from the left). Control (first one from the right) exhibits no red fluorescence.]]
+RFP has been largely produced in cells overexpressing tRNA<sup>Arg</sup>. No RFP can be observed in cells without rare tRNA overexpression.
+=='''Three Factor in Rare-Codon Switch'''==
+==Number of Rare Codons==
+In this part we want to explore the influence of the number of rare codons inserted in the mRNA. We have inserted 2, 4, 6, 8 AGG codons respectively after the start codon in luciferase gene.  T7 promoter or ''bla'' promoter<sup>[1]</sup> are used to control target protein mRNA amount. We use different combinations of number of AGG codons and strength of promoters to characterize regulation<sup>[1]</sup>.
+) ''bla'' promoter-luciferase (weaker promoter)
+A tandem of 2, 4, 6 or 8 AGG codons is inserted after the ATG codon of wild type luciferase
+Parts:
 *P''bla''-Luc-2AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567004 BBa_K567004])
@@ Line 27: / Line 60: @@
 *P''bla''-Luc-8AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567007 BBa_K567007])
+) T7 promoter-luciferase (stronger promoter)
+A tandem of 2, 4, 6 or 8 AGG codons is inserted after the ATG codon of wild type luciferase
+Parts:
 *PT7-Luc-2AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567008 BBa_K567008])
@@ Line 36: / Line 75: @@
 *PT7-Luc-8AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567010 BBa_K567010])
-'''Note:'''Luc for luciferase
-We have tested different combination of tRNA Modulators and Reporters and analyzed the influence of promoter strength for rare tRNA, number of rare codons in target protein mRNA and promoter strength for target protein gene respectively.
+[[image:11SJTU rare 12.jpg|center]]
-=='''The Working Curve of tRNA Modulator'''==
+===Influence of inserted AGG codon number===
-We have tested luciferin reaction in cells with different combinations of tRNA Modulators and Reporters. We examined the changes in luciferase enzyme activity over time after rare tRNA expression is induced. The amount of luciferase is reflected indirectly by the bioluminescence emitted from the luciferin reaction. Results are shown below:
+The influence of different number of rare codons in regulating protein biosynthesis is shown below:
-[[image:11-SJTU-compare-4.jpg|frame|center|''Fig.1'' (A)Enzyme activity of luciferase shown by bioluminescence emitted from the luciferin reaction reflecting the working curve of tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Arg</sup> ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) under Reporter P''bla''-Luc-4AGG（[https://parts.igem.org/wiki/index.php?title=Part:BBa_K567006 BBa_K567006]). (B)Enzyme activity of luciferase shown by bioluminescence emitted from the luciferin reaction reflecting the working curve of tRNA Modulator lacI-Ptrc-tRNA<sup>Arg</sup> ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) under Reporter PT7-Luc-4AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]). The working curve of tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Arg</sup> fits typical titration curve.]]
-Here we use the above two curves as examples to characterize the working curve of tRNA Modulator. Both curves fits typical titration curve, indicating that tRNA Modulator can function as a regulating tool.
+[[image:11SJTU_rare_11.jpg|center]|frame|center|''Fig.3'' (A) Comparing the influence of different number of rare codon insertions in luciferase production. P''bla''-Luc-4AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567005 BBa_K567005]), P''bla''-Luc-6AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567006 BBa_K567006]) and P''bla''-Luc-8AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567007 BBa_K567007]). (B) Comparing the influence of different number of rare codon insertions in luciferase production. Four Reporters are examined, including PT7-Luc-2AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567008 BBa_K567008]), PT7-Luc-4AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]), PT7-Luc-8AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567010 BBa_K567010]) and PT7-Luc-6AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567019 BBa_K567019]). ]]
-The rest of the working curves are shown here:
-<gallery caption="Pbla">
+This picture reflects more clearly that the more rare codons are inserted, the lower the background expression and the narrower the range of device regulation. We are able to predict the outcome of influence of different number of rare codons in protein biosynthesis, offering valuable information for device usage.
-image:11SJTUResultPlbla6.jpg|''Fig.2''
-image:11SJTUResultPbla8.jpg|''Fig.3''
-</gallery>
-<gallery caption="PT7">
+[[image:11sjtu_Compare_DATA_Fitting.png|frame|center|''Fig.4'' The comparison between background expression and induced expression of luciferase with different rare codon insertions. (A)PT7-luc reporters. (B) P''bla''-luc reporters]]
-image:11SJTUT7-2.png|''Fig.4''
-image:11SJTUT7-6.png|''Fig.5''
-image:11SJTUT7-8.png|''Fig.6''
-</gallery>
-'''Note''':Click to see large figures.
-From this experiment, we noticed that the typical working curve of tRNA Modulator can be better observed with IPTG induced tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Arg</sup> ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567001 BBa_K567001]) compared with UV excitation induced tRNA Modulator ''sulA'' promoter-tRNA<sup>Arg</sup>([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567002 BBa_K567002]) , though ''sulA'' promoter-tRNA<sup>Arg</sup> responded quicker to signals. We step further to test the influence of different Reporters with tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Arg</sup>.
+===Influence of different strengths of target protein promoters===
-=='''Target Protein mRNA Amount: regulated by different strength of target protein promoters, T7 promoter and bla promoter'''==
+We examined the influence of different Reporter promoters on the working curve of our device, which is reflected by luciferase activity. The working range of our device is pre-defined by the strength of target protein promoter, T7 promoter and ''bla'' promoter in our project.
-We examined the influence of different Reporter promoters on the working curve of tRNA Modulator, which is reflected by luciferase activity. Results showed that all the tRNA Modulator working curves fit titration curve, indicating that tRNA Modulator can act as a satisfying regulating tool.
+[[image:11sjtu_Compare_8.png|frame|center|''Fig.5'' (A) Luciferase (P''bla''-Luc-8AGG) production in cells overexpressing rare tRNA with ''lacI''-Ptrc-tRNA<sup>Arg</sup>. Luciferase production is reflected by bioluminescence emitted from the luciferin reaction. (B) Luciferase production in cells with PT7-Luc-8AGG as reporter. Here we analyze the influences of strong/weak promoter in luciferase production. Strong promoter (T7) of target gene can improve the titration curve, indicating that our device works better under strong target protein promoters. ]]
-[[image:11SJTUzhuzhuangtu-compare.jpg|frame|center|''Fig.7'' Enzyme activity of luciferase reaching plateau phase.]]
+==Location of Rare Codons==
-However, the working range of tRNA Modulator is pre-defined by the strength of target protein promoter, T7 promoter and ''bla'' promoter in our project.
+Experiment results showed that when there is 111bp interval between the two-copy 4AGG, background is lower than there is a 30bp interval. In another analysis, protein production can be induced to a higher level when there are two copies of AGG tandems.
-[[image:11sjtu_Compare_8.png|frame|center|''Fig.8'' (A) Working curve of tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Arg</sup> under Reporter P''bla''-Luc-8AGG reflected by bioluminescence emitted from the luciferin reaction. (B) Working curve of tRNA Modulator under Reporter PT7-Luc-8AGG. Here we analyze the influences of strong/weak promoter in the working curve of tRNA Modulator.
+[[image:11SJTU_rare_27.jpg|600px|frame|center|''Fig.6   A. When there is 111bp interval between the two-copy 4AGG, background is lower than there is a 30bp interval.     B. Protein production can be induced to a higher level when there are two copies of AGG tandems.'']]
-Moreover, strong promoter (T7) of target gene can improve the titration curve of tRNA Modulator, indicating that tRNA Modulator works better under strong target protein promoters. ]]
+'''Experiment results showed that luciferase tagged with two-copy 4AGG insertions with an interval of 111 bp can increase yield and lower background noise of target protein.''' We further conducted experiments to test its characters.
-=='''Influence of different numbers of AGG codons put after start codon ATG in tRNA Modulator Working Curve.'''==
+When we compare the two-copy 4AGG tagged luciferase with the single copy one, we find that two-copy 4AGG tagged luciferase has a higher yield.
-The working curve of tRNA Modulator still fit titration curve under Reporters with different number of AGG insertions, indicating that the number of rare codons in the Reporter will not affect the stability and function of the Modulator. The influence of different number of rare codons in regulating protein biosynthesis is shown below:
+[[image:11SJTU_rare_25.jpg|frame|center|''fig.7 Two-copy 4AGG tagged luciferase has a higher yield than single-copy'']]
-[[image:11sjtu_Compare_bla48_T72468.png|frame|center|''Fig.9'' (A) Comparing the influence of different number of rare codon insertions in tRNA Modulator working curve. Two Reporters are compared, P''bla''-Luc-4AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567005 BBa_K567005]) and P''bla''-Luc-8AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567007 BBa_K567007]). (B) Comparing the influence of different number of rare codon insertions in tRNA Modulator working curve. Four Reporters are examined, including PT7-Luc-2AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567008 BBa_K567008]), PT7-Luc-4AGG([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]), PT7-Luc-8AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567010 BBa_K567010]) and PT7-Luc-6AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567019 BBa_K567019]). ]]
+When we compare the two-copy 4AGG tagged luciferase with 8AGG tagged luciferase, we find that two-copy 4AGG tagged luciferase has a lower background.
-Results show that the more rare codons are inserted, the lower the background expression and the narrower the range of tRNA Modulator can regulate. Besides, since the number of rare codons will not affect system stability, it can be an excellent independent regulating factor.
-This picture reflects more clearly that the more rare codons are inserted, the lower the background expression and the narrower the range of tRNA Modulator regulation. We are able to predict the outcome of influence of different number of rare codons in protein biosynthesis, offering valuable information for device usage.
-=='''Future work'''==
-We have obtained PT7-Luc-2x4AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567021 BBa_K567021]) unexpectedly during our experiment. We have tested the enzyme activity of this part under tRNA Modulator control and have compared the curve with PT7-Luc-4AGG ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567009 BBa_K567009]) with the same tRNA Modulator. The result is shown below:
-[[image:11SJTUResultPT7_4_and_8(4-2).jpg|frame|center|''Fig.10'' the comparison of enzyme activity between PT7-Luc-4AG and PT7-Luc-2x4AGG under tRNA Modulator ''lacI''-Ptrc-tRNA<sup>Arg</sup>. ]]
-The result reflects the outstanding performance of the newly gained part. Two 4AGGs occurred in the beginning of the gene can bring lower background expression and much higher induced expression compared with luciferase with singe 4AGG insertion. The additional 4AGG does not affect the titration feature of the curve.
-Though we may not able to explain this phenomenon now, we have realized that exploring the potential regulating modes in our system is promising and significant. We will spare no effort in exploring and perfecting our system in our future work.
+[[image:11SJTU_rare_26.jpg|frame|center|''fig.8 Two-copy 4AGG tagged luciferase has a lower background than 8AGG tagged luciferase'']]
 =='''Related Biobrick'''==
@@ Line 116: / Line 130: @@
 PT7-Luc-8AGG         ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567010 BBa_K567010])
-PT7-Luc-2x4AGG       ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567021 BBa_K567021])
 PT7-RFP-6AGG         ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567017 BBa_K567017])
+PT7-Luc-2x4AGG(111bp)       ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567021 BBa_K567021])
+PT7-Luc-2x4AGG(30bp) ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567026 BBa_K567026])
+PT7-Luc-3x4AGG(30bp+111bp) ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K567027 BBa_K567027])
+===Reference===
+Ulrich Deuschlel., et al., ''Promoters of Escherichia coli: a hierarchy of in vivo strength indicates alternate structures'' The EMBO Journal vol.5 no. ''11'' pp.2987-2994, 1986