Kozak sequence

Cholera toxin B subunit CaMV 35S promoter/ nos terminator Kozak sequence, KDEL Lycopersicon esculentum Leaves 0.02% ofTSP Fruits 0.04% ofTSP 45... 

The tag sequence is amplified from plasmid pTA-His that provides a flexible linker and a double (His)g tag. The two amplicons can then be assembled and amplified by using an upstream T7 primer containing the promoter plus Kozak sequence and start codon for translation. A T-term/ for primer is used downstream to amplify across the tag and linker. The resulting construct contains the gene, the promoter, and the tag (Figure 6.26). This is key to the technology for the construct is then used for cell-free expression of the protein. 

The start codon is an AUG triplet designating methionine that is distinguished by the Kozak sequence, which base pairs with a portion of the 18S rRNA of the small subunit. 

Figure 15.2. Gene expression cassette. The drawing shows the features of the expression cassette. These inciude the promoter, 5 untransiated sequence, intron, Kozak sequence, therapeutic gene, 3 untrans-iated sequence, and the poiy-adenosine signai sequence.

Kozak sequence the base sequence that identifies the start codon in eukaryotic protein synthesis (12.5)... 

Once associated with mRNA, the pre-initiation complex scans downstream to locate a start codon (AUG). This process is driven by ATP, and requires the helicase activity of an elF. The start codon is recognised by base paring between the anti-codon on tRNA and the AUG on the mRNA. Codon-anticodon interaction is facilitated by yet more elFs. Usually, the first AUG codon that is encountered is used, especially if it is surrounded by the so called Kozak consensus sequence (named after its discoverer Marilyn Kozak). Occasionally a later AUG is used if the first AUG not in the context of a consensus Kozak sequence, or is very close to the 5 cap. This is in contrast to prokaryotes, where the AUG that will act as a start codon is located at the future P site by the position of the ribosome after base-pairing between the 16S rRNA of the ribosome and the Shine Dalgamo sequence. 

Formation of the SOS initiation complex. Recognition of the START codon (AUG) in mRNA is assisted by the preceding Kozak sequence (Table 71.3) before the pre-initiation complex is formed. Finally, the 60S ribosomal subunit combines with the 40S subunit to form the SOS initiation complex. 

Based on the analysis of 5 noncoding (5 NC) sequences from 699 vertebrate mRNAs, a consensus initiation sequence (also called Kozak sequence) has been identified as 5 -CC(A/G)CCAUGG—3, in which the nucleotides at —3 (Pu) and -1-4 seem to be particularly important (5). Database analysis of approximately 2600 vertebrate mRNAs provided further evidence for strong but not exactly identical trinucleotide biases at the —3, —2, and —1 positions as (A/G)NCAUG... 

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