7-methylguanosine triphosphate

Figure 35-10. The cap structure attached to the 5 terminal of most eukaryotic messenger RNA molecules. A 7-methylguanosine triphosphate (black) is attached at the 5 terminal of the mRNA (shown in blue), which usually contains a 2 -0-methylpurine nucleotide.

The primary transcripts generated by RNA polymerase II—one of three distinct nuclear DNA-depen-dent RNA polymerases in eukaryotes—are promptly capped by 7-methylguanosine triphosphate caps (Figure 35-10) that persist and eventually appear on the 5 end of mature cytoplasmic mRNA. These caps are necessary for the subsequent processing of the primary transcript to mRNA, for the translation of the mRNA, and for protection of the mRNA against exonucleolytic attack. 

Both ends of mRNA molecules are chemically modified. The 5 end is capped by 7-methylguanosine triphosphate. This modified guanine cap helps to protect the mRNA from attack by hydrolytic enzymes. Beyond that, the 7-methylguanosine triphosphate cap acts as a recognition element for ribosomes. The 3 end is modified by the addition of a poly A tail. This may consist of as few as 30 or as many as 200 adenine nucleotides. 

Expression of genetic information by transcription. [Note RNAs shown are eukaryotic.] me-7Gppp = 7-methylguanosine triphosphate cap, described on p. 414. AAA = poly-A tail, described on p. 414. 

Many more protein factors are involved in eukaryotic initiation some systems contain more than 10 initiation factors. Particular features of translation initiation are also different in the higher organisms. Most notably, prokaryotic ribosomes can initiate internally on an mRNA (even on circular RNAs), while in eukaryotes a preinitiation complex binds to the 5 -end of the mRNA and then progresses to an initiation complex. Eukaryotic mRNAs are capped at their 5 -end with a 7-methylguanosine triphosphate structure, and one of the eukaryotic initiation factors binds this capped end. The preinitiation complex then moves along the mRNA and initiates translation at the first AUG codon it comes to. Consistent with this scanning mechanism is the observation that eukaryotic mRNAs do not contain Shine-Dalgarno-like sequences. 

Sequence analysis of the 5 -ends of viral and nuclear mRNA molecules reveals that these are frequently capped , with an unusual structure in which 7-methylguanosine is joined by a (5 - 5 ) triphosphate link to a 2 -0-methyl nucleoside moiety which is the first residue in the (3 ->5 )-linked polynucleotide chain.166-168 The presence of this cap structure is required for ribosomal binding and translation to take place,167- 168 and 7-methylguanosine-5 -phosphate inhibits translation by preventing formation of the ribosome-mRNA complex.169... 

FIGURE 26-12 The 5 cap of mRNA. (a) 7-Methylguanosine is joined to the 5 end of almost all eukaryotic mRNAs in an unusual 5, 5 -triphosphate linkage. Methyl groups (pink) are often found at the 2 position of the first and second nucleotides. RNAs in yeast cells lack the 2 -methyl groups. The 2 -methyl group on the second nucleotide... 

Most eukaryotic mRNAs have a 5 cap, a residue of 7-methylguanosine linked to the 5 -terminal residue of the mRNA through an unusual 5, 5 -triphosphate linkage (Fig. 26-12). The 5 cap helps protect mRNA from ribonucleases. The cap also binds to a specific capbinding complex of proteins and participates in binding of the mRNA to the ribosome to initiate translation (Chapter 27). 

Structure of the 5 methylated cap of eukaryotic mRNA. A 7-methylguanosine (in red) is attached through a triphosphate linkage formed between its 5 -OH and the 5 -OH of the terminal residue in... 

In contrast to prokaryotic mRNAs, mRNAs from various eukaryotic cells and viruses have been found to contain a terminal 7-methylguanosine (m7G) residue linked from its 5 -position through a triphosphate bridge which was presented commonly as shown in the following Scheme 1. ... 

We first describe the synthesis of unsymmetrical a.y-dinucleoside triphosphates involving 7-methylguanosine. For the construction of the terminal cap structure, there might be two possible reaction modes where an activatable protecting group (X) was introduced into a nucleotide by direct displacement with phosphate hydroxyl group (Method A) and by pyrophosphorylation between Xp and pN (Method B). 

Attaches 7-methylguanosine to the penultimate nucleoside of RNA via a 5, 5 -triphosphate bridge. 

The second method for capping mRNA takes advantage of the activity of the vaccinia virus capping enzyme, also known as guanylyltransferase. This enzyme is commercially available. In the presence of GTP and S-adenosyl methionine (SAM), it can add a natural Cap structure (7-methylguanosine) to the 5 triphosphate of a RNA molecule. As it is an enzymatic reaction, it can bring a correct Cap to all mRNA molecules, and thus, it is optimal compared with in vitro transcription in the presence of standard Cap but similar theoretically to in vitro transcription in the presence of ARCA Cap. 

The m-RNA transcribed from DNA by RNA polymerase has a sequence which contains the information necessary for determining the whole amino acid sequence of the protein. Since a great many different proteins need to be synthesised, there are many thousands of different m-RNA molecules which have to be synthesised in the cell. Most chains of eukaryotic m-RNA are terminated at the 5 end by a cap of 7-methylguanosine which is linked to the chain by a triphosphate group. At the 3 end there is usually a poly A tail consisting of about 100 successive nucleotides with adenine (A) bases (11.129). 

Nucleoside triphosphatase [Unspecific diphosphate phosphohydrolase] (3.6.1.15) is produced and catalyzes the reaction NTP + H2O = NDP + orthophosphate. [Also hydrolyses other nucleoside triphosphates, diphosphate, thiamin diphosphate and FAD.] m7G (5 ) pppN pyrophosphatase [7-Methylguanosine,... 

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