Part:BBa_K1100000

ALeader

A kind of regulatory part that is induced by aminoglycoside drug.

Sequence and Features

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]

Background

Riboswitches are regulatory RNAs that regulate gene expression by binding small molecule metabolites. Recently, a novel riboswitch was reported which is present in the leader RNA of the aminoglycoside resistance genes that encode the aminoglycoside acetyl transferase (AAC) and aminoglycoside adenyl transferase (AAD) enzymes. When the aminoglycosides bind to the leader RNA, a conformational change will be induced, leading to the expression of the aminoglycoside resistance genes(Figure 1). (Jia X et al, 2013.) In addition, the leader RNA encompass an integrase site (attl1)which is overlapped by a short peptide encoded by an ORF (ORF11). ( Roth A et al.) Therefore, Aleader is a complicated riboswitch contains ORF11, cistron, aminoglycoside-binding domain and att1 recombination site. Due to the complicacy of Aleader, the secondary structure of it could not be predicted by most software such as Mfold and NUPACK. (Jia X et al, 2013.).

Figure 1. Induction of Aminoglycoside Resistance Genes by Leader RNA-Antibiotic Binding(Jia X et al, 2013.).

The 75nt RNA sequence of ALeader contains two SD sequences (ribosome binding sites) and an anti-SD sequence (CUUC) which can complementarily pair with either of the SD sequences. In the absence of aminoglycosides, anti-SD pairs with SD2. The binding of ribosomes to SD1 triggers the translation of a small peptide which stops at the stop codon ahead of SD2, therefore inhibits the translation of the gene after SD2. When aminoglycosides (kanamycin for example) exists, it will induce a structural change of Aleader. The anti-SD sequence pairs with SD1, consequently unmasking SD2 for ribosomal binding, which results in the translation of the following gene.

Our Aleader riboswitch demonstrates progressive induction of reporter genes in response to sublethal doses of the antibiotics. It makes sense because an antibiotic-resistant riboswitch must be able to detect low levels of antibiotics and activate the resistance mechanism before the cells are killed. Thus, Aleader turns out to be a novel translation regulatory part with high dynamic range, slight response delay and immense modification potential.

Mutation Design

One of the crucial tasks of synthesis biology is the modification or creation of bio-brick parts. Here, inspired by the riboswitch control of Aleader, we aimed to improve the parts’ sensitivity and precision in detection on different kinds of aminoglycoside antibiotics, by modifying the sequence of leader RNA and changing its structure. This part, hence, is a brand new inducible operator that could be regulated in translation, which might play an significant role in future biological researches.

Jia X et al. revealed the structural importance of leader RNA on the three loops. the SD1, SD2 and the anti-SD sequence(Figure 2). The yellow and white circles present the sites at which scission ability changes are induced with the addition of KanaB, which indicates the structural significance in these, like that the anti-SD would separate from SD2 and establish new interactions with SD1, ruling out the interaction with KanaB(Jia X et al, 2013). The other sites which decrease scission after the inducing of antibiotics are likely to have direct interaction with antibiotic moleculars. Mutations near these sites might change the parts’interaction capability with moleculars and thus interfere with the antibiotic specificity of inducing.

Figure 2.The Aminoglycosides Induce a Change in the Leader RNA Structure.

On the other hand, the binding affinity of anti-SD to the SD sites determines the signal noise ratio of the detection. A tighter binding would lead to a higher detection limit for the structural change while a more loosened binding would result in more false possive detections. Thus, mutations on the anti-SD site could regulate sensitivity of the part.

Take together, it is promising that mutational transformation on several interaction sites and anti-SD sites would to a great extend optimize our part.