Polypeptide chain termination

Scheme 10 Polypeptide chain termination by reaction with NCA anions...

Comment A simple test that is often done is to treat the lysate with 30 fiM EDTA prior to loading on the gradient, which leads to the disassembly of polysomes due to chelation of Mg2+ (Fig. 6.4B). EDTA is, however, not a very specific reagent and instead more specific drugs such as puromycin, which causes polypeptide chain termination and disaggregates polysomes, may be used. 

Polypeptide chain termination and stop codon readthrough on eukaryotic ribosomes... 

Stansfield 1, Tuite ME (1994) Polypeptide chain termination in Saccharomyces cerevisiae. Curr Genet 25 385-395... 

On the whole, our present understanding of archaeal translation is far from being complete. There is a considerable dearth of information on such essential aspects of archaeal biochemistry as the structure and sequences of the aminoacyl-tRNA synthethases, the mechanism of polypeptide chain termination and release, the number and complexity of the initiation factors the possibility that the archaea may resemble eucarya in having a more complex set of initiation factors than exists in bacteria is, in fact, suggested by the identification in archaea of hypusine-containing proteins (see section 2.4). The development of efficient and accurate cell-free systems using natural messenger RNAs is an obvious priority in order to elucidate these points. 

Premature stop codon polymorphisms, in which there is premature termination of the polypeptide chain by a stop codon (specific sequence of three nucleotides that do not code for an amino acid but rather specify polypeptide chain termination)... 

There are also codons for protein synthesis initiation (AUG) and polypeptide chain termination (UAG, UGA, and UAA). 

Mechanism of action of puromycin. Puromycin is able to enter the ribosome A site and function as an aminoacyl tRNA analogue, resulting in polypeptide chain termination in both... 

Causes premature polypeptide chain termination by binding to the A site... 

Polypeptides are linear polymers. One end of a polypeptide chain terminates in an amino acid residue that has a free —NHs group the other terminates in an amino acid residue with a free —C02 group. These two groups are called the N-terminal and the C-terminal residues, respectively ... 

Diagrammatic representation of translation on prokaryotic ribosomes. The elongation cycle starts by interaction of the 70S initiation complex with fMet- tRNA EFTu GTP. In all subsequent rounds of the cycle, fMet-tRNArEFT tGTP interacts with the mRNA ribosome complex carrying the growing polypeptide chain. Termination occurs when n amino acids have been incorporated, where n represents the number of codons between the initiation codon AUG and the termination codon (in this example UAA). 

Kaempfer, R., 1970, Dissociation of ribosomes on polypeptide chain termination and origin of single ribosomes. Nature 228 534. 

The early shut-off of protein synthesis apparently involves detachment of ribosomes from mRNA. The mRNA is apparently not massively degraded but is rendered non-functional (i.e., not translatable in an in vitro system). The detachment probably does not occur in the course of normal polypeptide chain termination since it is not prevented by cycloheximide. Either the detached ribosomes or others previously free are able to associate productively with viral mRNA. A specific, limited enzymic attack on cellular mRNA is a possibility as is an alteration in a recognition site on the ribosomes. Perhaps slightly more conceivable, but no better supported by evidence, is the suggestion that a change in the intracellular ionic environment might promote the detachment of cellular mRNA and the association of viral mRNA, as well as a change in the affinity of the DNA for RNA polymerase. 

Figure 4.5 The polypeptide chain of the enzyme pyruvate kinase folds into several domains, one of which is an a/p barrel (red). One of the loop regions in this barrel domain is extended and comprises about 100 amino acid residues that fold into a separate domain (blue) built up from antiparallel P strands. The C-terminal region of about 140 residues forms a third domain (green), which is an open twisted a/p structure.

Figure 6.12 (a) Schematic diagram of one subunit of GroEL. The polypeptide chain is folded info three domains. The equatorial domain (green) is the largest domain, comprising 10 a helices, and is built up from both the N-tetminal and the C-terminal regions. 

Figure 8.3 The DNA-binding protein Cro from bacteriophage lambda contains 66 amino acid residues that fold into three a helices and three P strands, (a) A plot of the Ca positions of the first 62 residues of the polypeptide chain. The four C-terminal residues are not visible in the electron density map. (b) A schematic diagram of the subunit structure. a helices 2 and 3 that form the helix-turn-helix motif ate colored blue and red, respectively. The view is different from that in (a), [(a) Adapted from W.F. Anderson et al., Nature 290 754-758, 1981. (b) Adapted from D. Ohlendorf et al., /. Mol. Biol. 169 757-769, 1983.]...

The polypeptide chain of the 92 N-terminal residues is folded into five a helices connected by loop regions (Figure 8.6). Again the helices are not packed against each other in the usual way for a-helical structures. Instead, a helices 2 and 3, residues 33-52, form a helix-turn-helix motif with a very similar structure to that found in Cro. 

The helices at the N-terminal regions of the two polypeptide chains are intertwined and make extensive contacts in the central part of the molecule to form a stable core. This core supports two "heads", each comprising the last three helices from one polypeptide chain. Alpha helix 3 in the middle of the subunit chain is quite long and forms the main link between the core and the head. 

Figure 8.21 Richardson-type diagram of the structure of one suhunit of the lac repressor. The polypeptide chain is arranged in four domains, an amino terminal DNA-hinding domain (red) with a helix-tum-helix motif, a hinge helix (purple), a large core domain which has two subdomains (green and hlue) and a C-terminal a helix. (Adapted from M. Lewis et al.. Science 271 1247-1254, 1996.)...

The polypeptide chain of the lac repressor subunit is arranged in four domains (Figure 8.21) an N-terminal DNA-hinding domain with a helix-turn-helix motif, a hinge helix which binds to the minor groove of DNA, a large core domain which binds the corepressor and has a structure very similar to the periplasmic arablnose-binding protein described in Chapter 4, and finally a C-terminal a helix which is involved in tetramerization. This a helix is absent in the PurR subunit structure otherwise their structures are very similar. 

Many biochemical and biophysical studies of CAP-DNA complexes in solution have demonstrated that CAP induces a sharp bend in DNA upon binding. This was confirmed when the group of Thomas Steitz at Yale University determined the crystal structure of cyclic AMP-DNA complex to 3 A resolution. The CAP molecule comprises two identical polypeptide chains of 209 amino acid residues (Figure 8.24). Each chain is folded into two domains that have separate functions (Figure 8.24b). The larger N-terminal domain binds the allosteric effector molecule, cyclic AMP, and provides all the subunit interactions that form the dimer. The C-terminal domain contains the helix-tum-helix motif that binds DNA. 

The side of the p sheet that faces away from DNA is covered by two long a helices. One of these helices contains a number of basic residues from the middle segment of the polypeptide chain while the second helix is formed by the C-terminal residues. Residues from these two helices and from the short loop that joins the two motifs (red in Figure 9.4) are likely candidates for interactions with other subunits of the TFIID complex, and with specific transcription factors. 

The polypeptide chain of p53 is divided in three domains, each with its own function (Figure 9.16). Like many other transcription factors, p53 has an N-terminal activation domain followed by a DNA-binding domain, while the C-terminal 100 residues form an oligomerization domain involved in the formation of the p53 tetramers. Mutants lacking the C-terminal domain do not form tetramers, but the monomeric mutant molecules retain their sequence-specific DNA-binding properties in vitro. 

Figure 9.22 Most tumorigenic mutations of pS3 are found in the regions of the polypeptide chain that are involved in protein-DNA interactions. These regions are loops L2 (green) and L3 (red) and a region called LSH (blue) which comprises part of p strand 9 as well as the C-terminal a helix.

The 12 residues between the second cysteine zinc ligand and the first histidine ligand of the classic zinc finger motif form the "finger region". Structurally, this region comprises the second p strand, the N-terminal half of the helix and the two residues that form the turn between the p strand and the helix. This is the region of the polypeptide chain that forms the main interaction area with DNA and these interactions are both sequence specific. 

The C-terminal transmembrane helix, the inner helix, faces the central pore while the N-terminal helix, the outer helix, faces the lipid membrane. The four inner helices of the molecule are tilted and kinked so that the subunits open like petals of a flower towards the outside of the cell (Figure 12.10). The open petals house the region of the polypeptide chain between the two transmembrane helices. This segment of about 30 residues contains an additional helix, the pore helix, and loop regions which form the outer part of the ion channel. One of these loop regions with its counterparts from the three other subunits forms the narrow selectivity filter that is responsible for ion selectivity. The central and inner parts of the ion channel are lined by residues from the four inner helices. 

The phosducin polypeptide chmn, of some 240 amino acids, is folded into two domains (Figure 13.16). The N-terminal domain is mostly a-helical and appears to be quite flexible since only a weak electron density is obtained in the structure determination. The actual path of the polypeptide chain from the end of helix to the beginning of helix Ba is tentative due to slight disorder. This region is close to serine 73 at the beginning of Ba, which also becomes disordered on phosphorylation. 

The polypeptide chain of Src tyrosine kinase, and related family members, comprises an N-terminal "unique" region, which directs membrane association and other as yet unknown functions, followed by a SH3 domain, a SH2 domain, and the two lobes of the protein kinase. Members of this family can be phosphorylated at two important tyrosine residues—one in the "activation loop" of the kinase domain (Tyr 419 in c-Src), the other in a short... 

C-terminal tail (Tyr 527 in c-Src). Phosphorylation of Tyr 419 activates the kinase phosphorylation of Tyr 527 inhibits it. Crystal structures of a fragment containing the last four domains of two members of this family were reported simultaneously in 1997—cellular Src by the group of Stephen Harrison and Hck by the group of John Kuriyan. The two structures are very similar, as expected since the 440 residue polypeptide chains have 60% sequence identity. The crucial C-proximal tyrosine that inhibits the activity of the kinases was phosphorylated in both cases the activation loop was not. 

Collagen chains are synthesized as longer precursors, called procollagens, with globular extensions—propeptides of about 200 residues—at both ends. These procollagen polypeptide chains are transported into the lumen of the rough endoplasmic reticulum where they undergo hydroxylation and other chemical modifications before they are assembled into triple chain molecules. The terminal propeptides are essential for proper formation of triple... 

The most remarkable feature of the antibody molecule is revealed by comparing the amino acid sequences from many different immunoglobulin IgG molecules. This comparison shows that between different IgGs the amino-terminal domain of each polypeptide chain is highly variable, whereas the remaining domains have constant sequences. A light chain is thus built up from one amino-terminal variable domain (Vl) and one carboxy-terminal constant domain (Cl), and a heavy chain from one amino-terminal variable domain (Vh), followed by three constant domains (Chi, Ch2. and Chs). 

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