Functional Analysis and Validation of Mg2+ Binding Sites of Intron-encoded Protein

doi:10.13560/j.cnki.biotech.bull.1985.2020-0012

Abstract

Abstract:

This work aims to screen and construct the mutants of having intron-encoded protein Mg²⁺ binding site,and to verify its effect on the “Retrohoming” efficiency of group II intron. Bioinformatics technology was used to screen key sites,site-directed mutation technology to construct mutants,and Targetron and blue-and-white spot counting methods to verify its “Retrohoming” efficiency. Results showed that 2 sites,D308 and D309,were identified as the core catalytic sites for group II intron-encoded protein Mg ²⁺ binding,and three mutants of these sites were successfully constructed,including two single-site mutants（D308A and D309A）and a double-site mutant（D308A/D309A）. The experimental results in Escherichia coli showed that all three mutants completely inactivated the “Retrohoming” function of group II intron. In conclusion,this work confirms that the Mg ²⁺ binding sites of group II intron-encoded protein is the core catalytic site of it playing function.

Key words: group II introns, intron-encoded protein, Mg²⁺ binding sites, Targetron, reverse transcription domain

CUI Gu-zhen, CHEN Xiang-hao, HONG Wei, ZHANG Zheng-rong, QI Ting-na, CHEN Zheng-hong. Functional Analysis and Validation of Mg²⁺ Binding Sites of Intron-encoded Protein[J]. Biotechnology Bulletin, 2020, 36(10): 165-172.

Figures/Tables 7

References 30

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Primers	Sequences（5'-3'）	Notes
lacZ-UF	CGGTCAATCCGCCGTTTGTTC	To detect the lacZ gene
lacZ-DR	GACCAGATGATCACACTCGG
IEP-F	ATCGAGGCTAGCGCTATATGCGTTGATG	To amplify the IEP fragment
IEP-R	CGTTCCAGATCTCCTTACTCGTA
D308A-F	TACGTCCGGTATGCGGCTGACTTCATTATCTCT	To mutate the D308A
D308A-R	AGCCGCATACCGGACGTATTTCAATACTTTATT
D309A-F	GTCCGGTATGCGGACGCTTTCATTATCTCTGTT	To mutate the D309A
D309A-R	AGCGTCCGCATACCGGACGTATTTCAATACTTT
D308A/D309A-F	TACGTCCGGTATGCGGCTGCTTTCATTATCTCTGTT	To mutate the D308A/D309A
D308A/D309A-R	AGCAGCCGCATACCGGACGTATTTCAATACTTTATT

Primers	Sequences（5'-3'）	Notes
lacZ-UF	CGGTCAATCCGCCGTTTGTTC	To detect the lacZ gene
lacZ-DR	GACCAGATGATCACACTCGG
IEP-F	ATCGAGGCTAGCGCTATATGCGTTGATG	To amplify the IEP fragment
IEP-R	CGTTCCAGATCTCCTTACTCGTA
D308A-F	TACGTCCGGTATGCGGCTGACTTCATTATCTCT	To mutate the D308A
D308A-R	AGCCGCATACCGGACGTATTTCAATACTTTATT
D309A-F	GTCCGGTATGCGGACGCTTTCATTATCTCTGTT	To mutate the D309A
D309A-R	AGCGTCCGCATACCGGACGTATTTCAATACTTT
D308A/D309A-F	TACGTCCGGTATGCGGCTGCTTTCATTATCTCTGTT	To mutate the D308A/D309A
D308A/D309A-R	AGCAGCCGCATACCGGACGTATTTCAATACTTTATT

	Strains and plasmids	Features	Source or reference
Strains	E. coli DH5α	Clone strain,FˉlacZΔM15Δ（lacZYA-argF）relA1	TaKaRa
	E. coli BL21（DE3）	F-,ompT,hsdS（rBB-mB）,gal,dcm（DE3）	TaKaRa
Plasmids	pMD19T	TA clone Vector,Ampr	TaKaRa
	pSY7	Derived from pSY6,lacI,T7 promotor,Amp^r	[24-25]
	pSY7-lacZ-635s	Derived from pSY7,targeting the sense strand 635 site of lacZ in BL21（DE3）	[25]
	pSY7-lacZ-1063a	Derived from pSY7,targeting the antisense strand 1063 site of lacZ in BL21（DE3）	[25]

	Strains and plasmids	Features	Source or reference
Strains	E. coli DH5α	Clone strain,FˉlacZΔM15Δ（lacZYA-argF）relA1	TaKaRa
	E. coli BL21（DE3）	F-,ompT,hsdS（rBB-mB）,gal,dcm（DE3）	TaKaRa
Plasmids	pMD19T	TA clone Vector,Ampr	TaKaRa
	pSY7	Derived from pSY6,lacI,T7 promotor,Amp^r	[24-25]
	pSY7-lacZ-635s	Derived from pSY7,targeting the sense strand 635 site of lacZ in BL21（DE3）	[25]
	pSY7-lacZ-1063a	Derived from pSY7,targeting the antisense strand 1063 site of lacZ in BL21（DE3）	[25]

Functional Analysis and Validation of Mg²⁺ Binding Sites of Intron-encoded Protein

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