+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Continuous in vitro evolution of bacteriophage RNA polymerase promoters



Continuous in vitro evolution of bacteriophage RNA polymerase promoters



Biochemistry. 33(39): 11980-11986



Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 008381515

Download citation: RISBibTeXText

PMID: 7522554

DOI: 10.1021/bi00205a037


Related references

Promoters recognized by Escherichia coli RNA polymerase selected by function: highly efficient promoters from bacteriophage T5. Journal of Bacteriology 164(1): 70-77, 1985

In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 1. RNA chain initiation, abortive initiation, and promoter escape at three bacteriophage promoters. Biochemistry 42(13): 3777-3786, 2003

Sequences of three promoters for the bacteriophage SP6 RNA polymerase. Nucleic Acids Research 14(8): 3521-3526, 1986

Interaction of RNA polymerase with promoters from bacteriophage fd. European Journal of Biochemistry 74(1): 107-113, 1977

Promoters for bacteriophage n 4 virion rna polymerase. Journal of Cellular Biochemistry Supplement (9 PART B): 150, 1985

Bacteriophage n 4 virion rna polymerase promoters. Calendar, R. Gold (ed.). Ucla (university Of California Los Angeles) Symposia On Molecular And Cellular Biology New Series, Vol. 30. Sequence Specificity In Transcription And Translation; Steamboat Springs, Colo., Usa, Apr. . Xxv 709p. Alan R. Liss, Inc: New York, N.y., Usa. Illus. 41-54, 1985, 1986

Sequences of three class II promoters for the bacteriophage T7 RNA polymerase. Journal of Molecular Biology 153(3): 825-830, 1981

Identification of bacteriophage K11 genomic promoters for K11 RNA polymerase. Journal of Biochemistry and Molecular Biology 35(6): 637-641, 2002

Differential binding of RNA polymerase to the pRM and pR promoters of bacteriophage lambda. Gene 23(2): 157-166, 1983

Sequence of a region near the left end of bacteriophage T3 DNA that contains three promoters for the E. coli RNA polymerase. Nucleic Acids Research 14(11): 4696-4696, 1986

The effect of a bacteriophage T4-induced polypeptide on host RNA polymerase interaction with promoters. Journal of Biological Chemistry 259(21): 13292-7, 1984

Locations and nucleotide sequences of three major class III promoters for bacteriophage T3 RNA polymerase on T3 DNA. Journal of Biological Chemistry 259(3): 1993-1998, 1984

Bacteriophage T7 early promoters: nucleotide sequences of two RNA polymerase binding sites. Journal of Molecular Biology 99(3): 419-443, 1975

In vitro transcription of pe38/polyhedrin hybrid promoters reveals sequences essential for recognition by the baculovirus-induced RNA polymerase and for the strength of very late viral promoters. Journal of Virology 72(4): 2991-2998, 1998