Ribosomal confirmation

The catalytic activities of the fortified wheat germ cell-free systems supplemented with each fraction were investigated (Fig. 2). As shown in Fig. 2, only 0 - 40 % ammonium sulfate fraction showed an enhancement in DHFR protein synthesis. This enhancement of protein experimental results and the fact that the various eukaryotic initiation factors are contained in synthesis was also confirmed by SDS-PAGE and autoradiography (Fig. 3). From the above 0-40 % ammonium sulfate fraction [5, 6], it can be concluded that the amount of initiation factors in a conventionally prepared wheat germ cell-fi extract is deficient for the translation of DHFR with internal ribosome entry site. Therefore, it needs to supplement a wheat germ cell-free extract with the fraction containing the limited initiation factors for the efficient protein translation, and this fortified cell-free system can be easily made by simple... 

In a typical experiment, paired reactions will be performed where either mRNA or RM are absent, to confirm that all translation products derive from the intended mRNA and reflect the activity of ER-bound ribosomes. As an additional control, mock translations can be performed and the membrane fraction removed by centrifugation (10 min, 60K, TLA100.2 rotor). The supernatant is then recovered, programmed with mRNA, and... 

The peptidyl transferase centre of the ribosome is located in the 50S subunit, in a protein-free environment (there is no protein within 15 A of the active site), supporting biochemical evidence that the ribosomal RNA, rather than the ribosomal proteins, plays a key role in the catalysis of peptide bond formation. This confirms that the ribosome is the largest known RNA catalyst (ribozyme) and, to date, the only one with synthetic activity. Adjacent to the peptidyl transferase centre is the entrance to the protein exit tunnel, through which the growing polypeptide chain moves out of the ribosome. 

Because the sequencing of DNA has become so straightforward, genes of several ribosomal proteins have been sequenced to allow an independent determination of the primary sequence of some of the proteins (Post et ai, 1979 Olins and Nomura, 1981). These studies have confirmed the sequences previously elucidated by protein chemical techniques. In the case of SI (the largest of all E. coli proteins), the combination of amino acid- and nucleotide-sequence determinations was used to provide the sequence (Schnier et ai, 1982). 

In principle the global structure of an RNA junction could be determined when it is complexed with bound protein. This has not been accomplished to date in RNA, but it has proven itself for DNA junctions. For example, the structure of DNA junctions bound by the junctionresolving enzymes T4 endonuclease VII (Pohler et al., 1996) and T7 endonuclease I (Declais et al., 2003) have both been determined by comparative gel electrophoresis. It was found that both proteins substantially alter the global shape of the DNA junction in different ways. These structures were both recendy confirmed by X-ray crystallography (Biertiimpfel et al., 2007 Hadden et al., 2007), showing that comparative gel electrophoresis functions reliably for protein complexes. There is no reason to expect that the method would not work equally well for RNA junction complexes. The binding of the ribosomal protein SI 5 to a three-way RNA junction has been studied by an electrophoretic approach that is related to comparative gel electrophoresis (Batey and Williamson, 1998). 

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