CAP

Eukaryotic mRNA has a 7-methyl-G cap (mCAP) at the 5' end. The cap protects the mRNA from nucleolytic degradation and recruits the ribosomal initiation complex to the start of the mRNA. Translation is dramatically enhanced by the presence of a cap. A cap can be incorporated in a transcription by including a mixture of cap analog and GTP (usually at a 4:1 ratio). 50% of the time, mCAP is inserted in the correct orientation to enhance translation. The other 50% of molecules are not substrates for efficient translation, reducing the specific activity of the transcript. More recently, Anti Reverse Cap Analog (ARCA) was introduced (1). ARCA can only insert in the proper orientation, leading to 100% activity.

5' Untranslated Region (5' UTR)

The 5' UTR does not encode for protein. However, these generally short regions can influence translational efficiency. The 5' UTR contains a sequence called the Kozak sequence which strongly influences translation efficiency. The optimal consensus Kozak sequence is GCCGCCRCCAUGG, where R is adenine or guanine. The AUG sequence is the site of translation initiation.

Open Reading Frame

The open reading frame of the mRNA is usually determined by the first AUG codon in the mRNA. Translation proceeds to the first in frame stop codon.

3' Untranslated Region (3' UTR) and Poly(A) Tail

The 3' UTR of an mRNA contains one or more polyadenylation sequences. In cells, the mRNA is cleaved at a polyadenylation sequence and a poly(A) tail is added to the end of the mRNA by poly(A) polymerase. The length of the poly(A) tail is an important determinant of translational efficiency and mRNA stability. When making mRNA in vitro by transcription, researchers commonly bypass the cleavage and polyadenylation step. Instead, a string of Ts is incorporated into the 3' end of the template and upon transcription a poly(A) tail is added opposite the Ts. It is desirable to have a long poly(A) tail, preferably 200 nucleotides or longer. Some natural 3' UTR sequences contain RNA instability elements that direct accelerated degradation of the mRNA. 3' UTRs are the main target for repression by microRNA. For applications where it is important to maintain normal regulation of the mRNA, it is desirable to utilize the endogenous 3' UTR of the transcript. For applications where maximal translation and duration of expression is desired, researchers commonly make chimeric mRNA that contains the 3' UTR of very stable transcripts such as alpha-globin or beta-globin.