Cap structure

In addition, for two coaxial armchair tubules, estimates for the translational and rotational energy barriers (of 0.23 meV/atom and 0.52 meV/atom, respectively) vvere obtained, suggesting significant translational and rotational interlayer mobility of ideal tubules at room temperature[16,17]. Of course, constraints associated with the cap structure and with defects on the tubules would be expected to restrict these motions. The detailed band calculations for various interplanar geometries for the two coaxial armchair tubules basically confirm the tight binding results mentioned above[16,17]. 

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.

As mentioned above, mammahan mRNA molecules contain a 7-methylguanosine cap structure at their 5 terminal, and most have a poly(A) tail at the 3 terminal. The cap stmcmre is added to the 5 end of the newly transcribed mRNA precursor in the nucleus prior to transport of the mELNA molecule to the cytoplasm. The S cap of the RNA transcript is required both for efficient translation initiation and protection of the S end of mRNA from attack by S —> S exonucleases. The secondary methylations of mRNA molecules, those on the 2 -hydroxy and the N of adenylyl residues, occur after the mRNA molecule has appeared in the cytoplasm. 

Biochemical and genetic experiments in yeast have revealed that the b poly(A) tail and its binding protein, Pablp, are required for efficient initiation of protein synthesis. Further studies showed that the poly(A) tail stimulates recruitment of the 40S ribosomal subunit to the mRNA through a complex set of interactions. Pablp, bound to the poly(A) tail, interacts with eIF-4G, which in turn binds to eIF-4E that is bound to the cap structure. It is possible that a circular structure is formed and that this helps direct the 40S ribosomal subunit to the b end of the mRNA. This helps explain how the cap and poly(A) tail structures have a synergistic effect on protein synthesis. It appears that a similar mechanism is at work in mammalian cells. 

The advantage of mRNA over plasmid transfection is the ability of in vitro transcription to allow precise control over features contained within the mRNA (Humphreys et al., 2005 Pillai et al., 2005 Westman et al, 2005). For example, mRNA can be prepared either with or without the physiological m7G(5/)ppp(5/)G cap structure and S poly(A) tail, which are important mediators of canonical translation initiation (Gallie, 1991 Hentze et al., 2006 Iizuka et al, 1994 Kahvejian et al, 2005 Tarun and Sachs, 1995). 

Example experiments using the previous methodologies are shown in Fig. 6.1. The major mRNA constructs described in this chapter are dia-grammatically represented in Fig. 6. IB and an example of in vitro transcribed and polyadenylated R-luc-4 sites mRNA is shown in Fig. 6.1A. In these experiments, translation of R-luc-4 sites mRNA is synergistically promoted by the physiological cap structure and the poly (A) tail (Fig. 6.1C), and full miR-dependent translational repression requires the presence of both modifications (Fig. 6.ID, Humphreys etal., 2005). (TheEMCV IRES-containing constructs are discussed later.)... 

In our work, we opted to deploy the IRES sequences within the 5 UTR of mono-cistronic reporter mRNA (Humphreys el al, 2005 Fig. 6.IB), which were directly transfected into HeLa cells. IRES-containing transcripts were further capped with the nonphysiological A(5/)ppp(5/)G cap structure,... 

The nature of the cap structure can also influence mRNA stability. The stability of mRNA capped with different ARCAs is determined in MM3MG cells that are electroporated with luciferase mRNAs containing a 60-nt poly(A) tract and different cap analogs. The experiment is done... 

Hsu, C., and Stevens, A. (1993). Yeast cells lacking 5 ->3 exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the S cap structure. Mol. Cell. Biol. 13, 4826-4835. 

Na salts of ribonucleotide triphosphates (Roche or Sigma) bovine serum albumin RNase-free, 20 mg/ml (Roche) RNasin ribonuclease inhibitor, 40 U/ml (Promega) both bacteriophage T7 RNA polymerase and RNA Cap structure analog m7G(5/)ppp(5/)G are from BioLabs DNase-RNase-free (Roche) complete EDTA-free proteinase inhibitors cocktail (Roche) pyruvate kinase (PK) (Roche). 

Oxidation of the cap structure To 400 fd of 32P-cap-labeled mRNA, 1.6 fd of 100 mMNaI04 is added and left on ice for 2 h, protected from light. The reaction is terminated by addition of 20 fd of 50% glycerol, passed through a G50 spin column and ethanol precipitated. The pelleted... 

A new imidazole-functionalized calix[4]arene ligand, able to form a dinuclear Cu2+ complex, has been reported to hydrolyze HPNP and ethyl p-nitropheny lphosphate [70]. The dinuclear complex was found to be 22-and 330-fold more reactive than the corresponding monomer towards the above substrates, respectively. Dinuclear Cu2+ complexes of linked triazacyclononane ligands are reported to promote the hydrolysis of the monoribonucleotide GpppG, a model for the 5 -cap structure of mRNA [71]. The dinuclear complexes offer some 100-fold higher reactivity compared to the mononuclear Cu2+-triazacyclononane system. 

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... 

A 7-methylguanosine cap is added to the 5 end while the RNA molecule is still being synthesized. The cap structure serves as a ribosome-binding site and also helps to protect the mRNA chain from degradation. 

The small ribosomal subunit binds to the mRNA. In prokaryotes, the 16S rRNA of the small subunit binds to the Shine-Dalgamo sequence in the 5 untranslated region of the niRNA. In eukaryotes, the small subunit binds to the 5 cap structure and slides down the message to the first AUG. 

Fig. 1.44. Modifications at the 5 and 3 ends of eucaryotic mRNA. Eucaryotic mRNAs possess a cap structure" at their 5 ends and a 100-200 base long poly-A tail at their 3 ends.

Capping at the 5 -end of the pre-mRNA occurs immediately after incorporation of about 30 nucleotides in the primary transcript. The 5 cap structure is required for the binding of the mRNA to the 40S subunit of the ribosome during the initiation of translation. Capping is also ascribed a stabilizing function for mRNA. 

The fimction of eIF-2 is illustrated schematically in Fig. 1.55. eIF-2 belongs to the superfamily of regulatory GTPases (see ch. 5). elF-2 fulfills the task of bringing the methionyl-initiator-tRNA to the 40S subimit of the ribosome. The active eIF-2 GTP form binds the methionyl-initiator-tRNA, associates with the cap structure of the mRNA, then commences to scan along the mRNA. Once an AUG codon is encoimte-red, the boimd GTP is hydrolyzed to GDP, resulting in the dissociation of the... 

Fig. 1. 55. The function of eIF-2 in eucaryotic translation. eIF-2, the initiator protein for the translation is a regulatory GTPase that occurs in an active GTP-form and in an inactive GDP form (see ch. 5). The active eIF-2 GTP forms a complex with the initiator-tRNA, fMet-tRNA "" and the 40S subunit of the ribosome. This complex binds to the cap structure of mRNA to initiate the scanning process. eIF-2 undergoes an activation cycle typical for regulatory GTPases the inactive eIF-2 GDP fom is activated with the assistance of the eIF-2B protein into the active elF-2 GTP form. eIF-2B acts as a G-nucleotide exchange factor in the cycle (see ch. 5).

---