RNA

Part:BBa_I1013:Design

Designed by: June Rhee, Connie Tao, Ty Thomson, Louis Waldman   Group: Antiquity   (2003-01-31)


CI(1) IS10 asRNA


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Complementary to beginning of BBa_I1010 transcript covering RBS, start codon, and 73 bp into coding sequence. Secondary structure designed for the IS10 anti-sense mRNA mechanism (See below).
Anti-sense

The success of this system clearly rests on the ability to effectively and specifically target mRNA transcripts for degradation using anti-sense RNA. While many papers, articles, and books have been written on the subject, there are no consensus anti-sense building strategies presented. We thus chose to implement three different types of antisense inhibition: KISS, micRNA, and IS10. In the description that follows, the following nomenclature will be used:

target- the mRNA transcript that we wish to inhibit.

anti-sense- the anti-sense molecule which will bind and inhibit target.

IS10 <img src="http://biobricks.ai.mit.edu/IAP_Projects/YoungPower/as_IS10.gif">

This method is modeled after the mechanism by which IS10 inhibits production of IS10 transposase. The anti-sense strand is transcribed from the complementary strand of the target (see below), resulting in an anti-sense strand that is 115 bp long, of which 35 bp are complementary to the target. In the absense of a target, these 35 bp form a weak stem loop with the rest of the anti-sense molecule (see below). The key element of the system is the loop at the tip of this stem loop (C-G-G-C-U-U...), which is held in a linear state by the rest of the loop. Upon exposure to the target, the linear loop is able to bind to the 5' end of the target (G-C-C-G-T-T...), and initiate an energetically-favorable zipping/twisting-together of the target and the 5' end of the stem loop (see below). In other words, one side of the weakly stable anti-sense stem loop binds 35 bp of the target, to form a more stable duplex.

I1010 and I1013

Biobricks part BBa_I1013 codes for the exact anti-sense stem loop used in IS10, with two base changes. The 5'-most residues from IS10 anti-sense transcript ( U-C), which do not form part of the stem loop, were changed to G-A. These two bases are reverse-complementary to the first two base pairs of the wildtype cI coding region of BBa_I1010, and thus can bind this region. The rest of the stem loop is wild-type.

The BBa_1010 transcript is targeted by BBa_I1013. The first 35 bases at the 5' end of BBa_I1010 are identical to the first 35 bases at the 5' end of the wild type target, with two differences. Note that three bases T-G-C (which code for cysteine) have been inserted at the 5' end of the cI coding region immediately after the start codon. This allows us to use a wild-type binding pattern at the base of the stem. Since this cysteine is added to the N-terminus of cI, it is not expected to alter the repression ability of cI.

References

References (unparsed) here:

Case, C., Roels, S., Jense, P., Lee, J., Kleckner, N. and Simons, R. (1989). The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain. EMBO 8(13): 4297-4305.

Jain, C. (1995). IS10 Antisense Control in Vivo is Affected by Mutations Throughout the Region of Complementarity Between the Interacting RNAs. J. Mol. Biol. 246:585-594.

Jain, C. (1997). Models for Pairing of IS10 Encoded Antisense RNAs in vivo. J. theor. Biol. 186: 431-439.

Kittle, J.D., Simons, R.W., Lee, J., and Kleckner, N. (1989). Insertion Sequence IS10 Anti-sense Pairing Initiates by an Interaction Between the 5' End of the Target RNA and a Loop in the Anti-sense RNA. J. Mol. Biol. 210:561-572.

Lutz, R., and Bujard, H. (1997). Independent and tight regulation of transcriptional units in E. coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research 25(6): 1203-1210.

Ma, C., and Simons, R. (1990). The IS10 antisense RNA blocks ribosome binding at the transposase translation initiation site. EMBO 9(4):1267-1274.

E. coli codon usage table (<a href="http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html">http://bioinfo.weizmann.ac.il:3456/kegg/codon_table/codon_eco.html</a>).

Source

Custom design.

References