Ribosome active sites

Wimberly BT, Guymon R, McCutcheon JP, White SW, Ramakrishnan V (1999) A detailed view of a ribosomal active site the structure of the LI 1-RNA complex. Cell 97 491-502... 

Proteins are assembled on ribosomes in a linear sequence specified by the base sequence in an mRNA. If any of the amino acids is not present, the process stops when the base sequence that specifies its incorporation into the protein enters the ribosomal active site. 

Macrolides are a group of antibiotics, produced in nature by many actinomycetes strains, that are composed of a 12- to 16-membered lactone ring, to which one or more sugar substituents is attached. They target the peptidyl transferase center on the 50S ribosomal subunit and function primarily by interfering with movement of the nascent peptide away from the active site and into the exit tunnel. 

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 the following section, we describe protocols for tests aimed at screening for compounds capable of interfering with some of the main activities of this factor, such as (a) recognition and binding of initiator tRNA (b) codon-dependent ribosomal binding of fMet-tRNA leading to the formation of a 30S or 70S initiation complex (c) ribosome-dependent hydrolysis of GTP and (d) accommodation of fMet-tRNA in the ribosomal P-site and formation of the first peptide bond (initiation dipeptide formation). 

It has become apparent that the method of crosslinking can dramatically affect the activity of an immunotoxin in vivo. This is true not only with regard to possible direct blocking by the crosslinker of the enzymatic active site which is responsible for inactivation of ribosomes, but the chemistry of conjugation also is an important factor in proper binding and entry of the... 

The large ribosomal subunit at 2.4 A resolution. (A) The particle rotated with respect to the crown view so that its active site cleft can be seen. (B) The crown view. (C) The back view of the particle, i.e., the crown view rotated 180° about its vertical axis. Reprinted with permission from Ban et al., Science 289, 905 (2000). Copyright 2000 American Association for the Advancement of Science. 

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. 

Fig. 4.1. Fundamentals of the ubiquitin system. Adapted from Ref [5]. Figure 4.1 shows the fundamentals of the ubiquitin system. (1) Ubiquitin is synthesized in linear chains or as the N-terminal fusion with small ribosomal subunits that are cleaved by de-ubiquitylating enzymes to form the active protein. Ubiquitin is then activated in an ATP-dependent manner by El where a thiolester linkage is formed. It is then transthiolated to the active-site cysteine of an E2. E2s interact with E3s and with substrates and mediate either the indirect (in the case of HECT E3s) or direct transfer of ubiquitin to substrate. A number of factors can affect this process. We know that interactions with Hsp70 can facilitate ubiquitylation in specific instances and competition for lysines on substrates with the processes of acetylation and sumoylation may be inhibitory in certain instances. (2) For efficient proteasomal targeting to occur chains of ubiquitin linked internally through K48 must be formed. This appears to involve multiple...

Peptide bond formation is the essential reaction catalyzed by the ribosome. Despite its importance, it was for a long time not the focus of ribosomal research, for several reasons. First, before the determination of the high-resolution ribosome crystal structures almost nothing was known about the active site. Second, under most experimental conditions accommodation of the incoming aminoacyl-tRNA is rate limiting for peptide bond... 

The origin of the idea that a ribosome might be a ribozyme is derived from the experiment in which peptidyl transferase activity was observed even after digestion of protein components of the ribosome [15]. This was surprising because the most important biological function involved in the synthesis of proteins is catalyzed by RNA. Recently, a large ribosomal subunit from Haloarcula marismortui was determined at a resolution of 2.4 A [16, 155]. Importantly, because of the absence of proteins at the active site, it was concluded that the key peptidyl transferase reaction is accomplished by the ribosomal RNA (rRNA) itself, not by proteins. How does it work ... 

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