kozak序列
The Kozak consensus sequence was originally defined as ACCAUGG following an analysis of the effects of single mutations surrounding the initiation codon (AUG) on translation of the preproinsulin gene (1). Subsequent mutagenesis studies and a survey of 699 vertebrate mRNAs extended the consensus sequence for translation initiation to GCCGCCACCAUGG, where the A in the underlined AUG start codon is coordinate 1 and the A at position -3 could also be a G (2,3). Functional studies on preproinsulin and alpha-globin translation in cells indicated that a purine (usually A) in position -3 is crucial for efficient initiation of translation, and in its absence, a G at position +4 is essential (4). RNA templates with CCACCAUGG were translated well in a study using the Rabbit Reticulocyte Lysate System(a,b,c) (Cat.# L4960) in vitro translation reaction (5). In the in vitro translation
reaction
using
the
TNT?/sup>
T7
Quick
Coupled
Transcription/Translation System(a,b,c,d,e) (Cat.# L1170, L1171, L5540), PCR products can be used as templates
. The upstream primer for generating the
PCR template was designed to contain the T7 promoter for transcription by T7 RNA Polymerase (Cat.# P2075) and the Kozak consensus sequence (CCACCAUG or CCACCAUGG) for efficient translation
.
References
Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283.
Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947.
Kozak, M. (1987) An analysis of 5?noncoding sequences from 699 vertebrate messenger RNAs. Nucl. Acids Res. 15, 8125.
Kozak, M. (19) The scanning model for translation: an update. J. Cell Biol. 108, 229.
Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucl. Acids Res. 18, 2828.
Hogervorst, F.B.L. (1997) The protein truncation test (PTT). Promega Notes 62, 7.
Kozak 序列对于哺乳动物细胞表达的设计非常重要，但有时为引起足够重视
Ribosomal Binding Site Sequence Requirements
Protein synthesis is regulated by the sequence and structure of the 5' untranslated region (UTR) of the mRNA transcript. In prokaryotes, the ribosome binding site (RBS), which promotes efficient and accurate translation of mRNA, is called the Shine-Dalgarno sequence after the scientists that first described it. This purine-rich sequence of 5' UTR is complementary to the UCCU core sequence of the 3'-end of 16S rRNA (located within the 30S small ribosomal subunit). Various
Shine-Dalgarno sequences have been found in prokaryotic mRNAs (see Figure 1 for the consensus sequence). These sequences lie about 10 nucleotides upstream from the AUG start codon. Activity of a RBS can be influenced by the length and nucleotide composition of the spacer separating the RBS and the initiator AUG.
In eukaryotes, the Kozak sequence A/GCCACCAUGG, which lies within a short 5' untranslated region, directs translation of mRNA. An mRNA lacking the Kozak consensus sequence may be translated efficiently in Ambion's in vitro systems if it possesses a moderately long 5' UTR that lacks stable secondary structure. Our data demonstrate that in contrast to the E. coli ribosome, which preferentially recognizes the Shine-Dalgarno sequence, eukaryotic ribosomes (such as those found in retic lysate) can efficiently use either the Shine-Dalgarno or the Kozak ribosomal binding sites.
Figure 1. Consensus RBS Sequences. The +1 A is the first base of the AUG initiator codon (shaded) responsible for binding of fMet-tRNAfMet. The underline indicates the ribosomal binding site sequence, which is required for efficient translation.
在真核细胞表达过程中，KozaK序列是保证目的基因转录产物能够被翻译的重要影响因素。Kozak在19年的一项研究中表明，真核核糖体进行翻译的共有序列是：G C C G C C A-3／G C C A 1 T G G +4。但是Kozak指出，在实际操作中，只需考虑－3和＋4位置即可判断起始密码子的强弱，只要－3位置出现嘌呤（ A或G ），那么若其它位
置出现偏离共有序列的情况只会轻微影响起始效率；如果－3位置没有出现嘌呤（A／G），那么在+4位置上的G就成为进行有效翻译所必不可少的条件。
一般基因天然都有Kozak序列的存在，克隆ORF时向前移四个碱基就好了。
做克隆时，按Kozak序列改造一下引物就可以了。
所谓Kozak规则，即第一个ATG侧翼序列的碱基分布所满足的统计规律，若将第一个ATG中的碱基A，T，G分别标为1，2，3位，则Kozak规则可描述如下：
(1)第4位的偏好碱基为G；
(2)ATG的5’端约15bp范围的侧翼序列内不含碱基T；
(3)在-3，-6和-9位置，G是偏好碱基；
(4)除-3，-6和-9位，在整个侧翼序列区，C是偏好碱基。
Kozak规则是基于已知数据的统计结果，不见得必须全部满足，一般来说，满足前两项即可。
