Polypeptide chain termination ribosomes

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

The previous sections have introduced the major participants in protein synthesis—mRNA, aminoacylated tRNAs, and ribosomes containing large and small rRNAs. We now take a detailed look at how these components are brought together to carry out the biochemical events leading to formation of polypeptide chains on ribosomes. Similar to transcription, the complex process of translation can be divided into three stages—initiation, elongation, and termination—which we consider in order. We focus our description on translation in eukaryotic cells, but the mechanism of translation is fundamentally the same in all cells. 

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... 

Translation converts the genetic information embodied in the base sequence (codon) of mRNA into the amino acid sequence of a polypeptide chain on ribosomes. Protein biosynthesis (Amstein and Cox, 1992 Lee and Lorsch, 2(X)4 Moldave, 1981) begins with the activated amino acids (aminoacyl-tRNA) and is characterized by three distinct phases initiation, elongation and termination. 

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). 

In E. coli, mutation of a given triplet to a UAG or UAA sequence leads to the premature termination of the polypeptide chains. These data and many other genetic studies led to the conclusion that UAG and UAA constitute termination codons in E. coli. Once such codons have been reached in the course of translation of a messenger RNA ribbon, a molecular mechanism is put in gear which releases the messenger from ribosomes, detaches the polypeptide chain from ribosomes and the tRNA that carries the carboxyl terminus, and releases the remaining tRNA from ribosomes. 

Puromycin has a structure closely analogous to the 3 terminus of a tyrosine-tRNA, and it inhibits protein synthesis by accepting, in place of an aminoacyl-tRNA, an incomplete polypeptide chain from ribosome-bound peptidyl-tRNA, thus prematurely terminating protein synthesis. We have found that irradiation at 2537 A (low-pressure Hg lamp) of solutions containing puromycin and ribosomes from E. coli leads to significant covalent incorporation of puromycin into the ribosome. For... 

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. 

Termination Three codons (UAA, UAG and UGA) are stop codons which do not code for any amino acid but, instead of attaching to a tRNA molecule, they bind a protein release factor. When one of these factors is encountered by the ribosome, peptidyl transfer is aborted, the completed polypeptide chain released by hydrolysis and the ribosome subunits separate. The N-terminal methionine unit is then removed from the polypeptide chain. 

Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JH, Noller HF (2001) Crystal structure of the ribosome at 5.5A resolution. Science 292 883—896 Zhouravleva G, Frolova L, Le Goff X, Le Guellec R, Inge-Vechtomov S, Kisselev L, Philippe M (1995) Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRFl and eRF3. EMBO J 14 4065-4072... 

Stags 4 Termination and Release Completion of the polypeptide chain is signaled by a termination codon in the mRNA. The new polypeptide is released from the ribosome, aided by proteins called release factors. 

A large number of components are required for the synthesis of a polypeptide chain. These include all the amino acids that are found in the finished product, the mRNA to be translated, tRNAs, functional ribosomes, energy sources, and enzymes, as well as protein factors needed for initiation, elongation, and termination of the polypeptide chain. 

Simplicity argues that the genetic blueprint specifying amino acid sequences in proteins should consist of consecutive, nonoverlapping triplets. This assumption turned out to be correct, as is illustrated by the DNA sequence for a gene shown in Fig. 5-5. In addition to the codons that determine the sequence of amino acids in the protein, there are stop codons that tell the ribosomal machinery when to terminate the polypeptide chain. One methionine codon serves as an initiation codon that marks the beginning of a polypeptide sequence. One of the valine codons sometimes functions in the same way. 

How are the possible choices for newly formed proteins made Much seems to depend upon the amino acid sequences at the ends of the polypeptide chains. As they emerge from a ribosome, some N-terminal signal sequences bind to recognition proteins. One such protein labels the ends of proteins destined for secretion into the vesicles of the ER. This protein ensures that the protein end binds to the signal recognition particle (SRP), enters a translocon pore, and undergoes cotranslational passage into the periplasmic space in bacteria or the ER in eukaryotes. Cotranslational modification reactions also occur both in the... 

Initiation factors contribute to the ribosome complex with the messenger RNA and the initiator methionyl-tRNA. Elongation factors assist the binding of all the other tRNAs and the translocation reaction that must occur after each peptide bond is made. Termination factors recognize a stop signal and lead to the termination of polypeptide synthesis and the release of the polypeptide chain and the messenger from the ribosome. 

Termination is triggered when the ribosome reaches a stop codon on the mRNA. At this stage, the polypeptide chain is released and the ribosomal subunits dissociate from the mRNA. Various protein factors are involved in all three phases of protein biosynthesis. 

A protein tail, which is the same in all library members, is fused to the C-terminus of the ribosome display construct and serves as a spacer. This spacer has two main functions. First, it tethers the synthesized protein to the ribosome. Second, it keeps the structured part of the protein outside the ribosome and allows its folding and interaction with ligands, without clashing with the ribosomal tunnel. The ribosomal tunnel covers between 20 and 30 C-terminal amino acids of the nascent polypeptide chain during protein synthesis and can therefore prevent the folding of the protein (Malkin and Rich, 1967 Smith et al., 1978). 

The molecular mechanism of translation in eukaryotes is very similar to that in bacteria. The activation of amino acids and attachment to tRNAs and the steps of initiation, elongation, and termination of polypeptide chains are essentially the same in overall terms. The small and large ribosomal subunits of bacteria and eukaryotes are equivalent with respect to their roles in initiation and elongation of chains. 

At the ribosome, which travels along the mRNA, the tRNA molecule is bound such that its anticodon can interact with a nucleotide triplet on mRNA (the codon). If the anticodon is complementary to a codon triplet on the mRNA, the amino acid attached at the 3 -terminus of the tRNA is transferred to the amino terminus of the growing polypeptide chain if it is not complementanty, the tRNA is rejected and another one is checked for complementary. The whole process is repeated until the synthesis of the protein is completed. It is initiated, as well as terminated, by specific codons regulating this translation. 

The N-terminal a-NH2 group appears to be involved in more modification than does the C-terminus. Acetylation, formylation and methylation are three quite common N-terminal derivatives, and just like in the case of the C-terminal end, amino acids can be added to the N-terminus of the finished polypeptide chain in the absence of ribosomes, but in this case the donor is amino acyl-tRNA. 

Once protein synthesis is initiated, amino acids are added to the peptide chain corresponding to each triplet in the mRNA until the ribosome encounters a termination or stop codon, whereupon the polypeptide chain is released from the ribosome, and assumes its final configuration. A ribosome covers about 50 bases of an mRNA, which is usually hundreds of bases long. Thus, several ribosomes translate an mRNA consecutively and simultaneously at any instant as shown in Fig. 2.4. A group of ribosomes translating a message is called a polyribosome . 

The final phase of translation is termination. How does the synthesis of a polypeptide chain come to an end when a stop codon is encountered Aminoacyl-tRNA does not normally bind to the A site of a ribosome if the codon is UAA, UGA, or UAG, because normal cells do not contain tRNAs with anticodons complementary to these stop signals. Instead, these stop codons are recognized by release factors (RFs), which are proteins. One of these release factors, RFl, recognizes UAA or UAG. A second factor, RF2, recognizes UAA or UGA. A third factor, RF3, another G protein homologous to EF-Tu, mediates interactions between RFl or RF2 and the ribosome. 

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