Terminator codons

An opening frame contains a series of codons (base triplets) coding for amino acids without any termination codons. There are six potential reading frames of an unidentified sequence. 

The terms first, second, and third nucleotide refer to the individual nucleotides of a triplet codon. U, uridine nucleotide C, cytosine nucleotide A, adenine nucleotide G, guanine nucleotide Term, chain terminator codon. AUG, which codes for Met, serves as the initiator codon in mammalian cells and encodes for internal methionines in a protein. (Abbreviations of amino acids are explained in Chapter 3.)... 

The charging of the tRNA molecule with the aminoacyl moiety requires the hydrolysis of an ATP to an AMP, equivalent to the hydrolysis of two ATPs to two ADPs and phosphates. The entry of the aminoacyl-tRNA into the A site results in the hydrolysis of one GTP to GDP. Translocation of the newly formed pep-tidyl-tRNA in the A site into the P site by EF2 similarly results in hydrolysis of GTP to GDP and phosphate. Thus, the energy requirements for the formation of one peptide bond include the equivalent of the hydrolysis of two ATP molecules to ADP and of two GTP molecules to GDP, or the hydrolysis of four high-energy phosphate bonds. A eukaryotic ribosome can incorporate as many as six amino acids per second prokaryotic ribosomes incorporate as many as 18 per second. Thus, the process of peptide synthesis occurs with great speed and accuracy until a termination codon is reached. 

The mRNA is read continuously from a start codon (AUG) to a termination codon (UAA, UAG, UGA). 

Fig. 8.4 Outline of the main events in protein synthesis initiation, elongation, translocation and termination. AUG is an initiation codon on the mRNA it codes for Af-fomiylmelhionine and initiates the formation of the 70S rihosome. UAG is a termination codon it does not code for any amino acid and brings about termination of protein synthesis.

Sachs, M. S., Wang, Z., Gaba, A., Fang, P., Belk, J., Ganesan, R., Amrani, N., and Jacobson, A. (2002). Toeprint analysis of the positioning of translation apparatus components at initiation and termination codons of fungal mRNAs. Methods 26, 105-114. 

EGF (synthetic gene) CaMV 35S promoter/ nos terminator Codon-optimized for high level peptide expression in Escherichia coli N. tabacum (leaves) 0.001% ofTSP 38... 

PETRACEK, M.E., NUYGEN, T., THOMPSON, W.F., DICKEY, L.F., Premature termination codons destabilize ferredoxin-1 mRNA when ferredoxin-1 is translated, Plant J., 2000,21,563-569. 

A gene encodes a protein with 150 amino adds. There is one intron of 1000 bps, a 5 -untranslated region of 100 bp and a 3 -nntranslated re on of 200 bp. In the final processed mRNA, how many bases lie between the start AUG codon and the final termination codon ... 

Fig.1. The in vitro site-directed suppression mutagenesis system. Utilizing site-directed mutagenesis a specific codon within a gene under the control of an inducible promoter is converted to an amber termination codon. The gene is added to a cell-free expression system and transcription is induced in the presence of an aminoacyl suppressor tRNA, yielding protein containing the noncoded amino acid at the site corresponding to the termination codon...

During the normal process of termination of translation, stop codons are recognized by protein release factors (RF). Although the details of the process are not fully understood, it is believed that when a termination codon reaches the ribosomal A-site, the RF associates with the ribosomal-mRNA complex, inducing the peptidyl-transferase center to hydrolyze the ester bond of the pepti-... 

Competition with release factors can be reduced by deactivating a release factor in a cell-free lysate prepared from a prokaryote. This option is not available in rabbit reticulyte lysate since mammals have only two release factors (RFs), termed eukaryotic release factors 1 and 3 (eRFl and eRF3). In eukaryotes, eRFl recognizes all three stop codons and eRF3 stimulates eRFl activity in the presence of GTP [41]. Deactivation of either RF would effectively deactivate all three stop codons, and at least one termination codon must function properly for the suppression method to be useful. 

Deactivation of an RF is feasible, however, in prokaryotes. In E. coli, three RFs are involved in the termination process, and there is a degree of codon specificity for two of them. RFl recognizes the UAG and UAA stop codons, while RF2 recognizes the UGA and UAA stop codons [16, 42]. RFS, which is not codon specific, stimulates the activity of the other two. Thus, if RF 1 is deactivated, UAA and UGA remain functional termination codons, but UAG should no longer signal for termination of protein synthesis. To test this hypothesis, the Chamberlin lab investigated a mutant strain of E. coli that produces a faulty RFl [29]. 

Building on earlier work of Osawa and co-workers [55], Oliver and Kowal [52] tested the feasibility of introducing a noncoded amino acid at an unassigned codon in M. luteus. DNA templates were prepared which coded for 19-mer polypeptides containing either the unassigned codon AGA(Arg) or the termination codon TAG at position 13 under the control of a T7 RNA polymerase promoter. The corresponding tRNAs, produced as described in Sect. 2, were based on tRNA and acylated with phenylalanine. The tRNA was modified to prevent recognition by the alanine aminoacyl-tRNA synthetase and to increase translational efficiency. 

A suppressor tRNA corresponding to the termination codon TGA was also tested, but... 

The final step in protein biosynthesis is chain termination. Natural mRNA molecules contain termination codons UAA, UGA, or UAG There are no tRNAs that have anticodons which are complementary to these codons. When the growing peptide chain encounters one of these termination codons, the peptidyl-tRNAis transferred to water instead of another aminoacyl-tRNA. The peptidyl-tRNA is hydrolyzed to free the completed protein and the tRNA. Chain termination completes protein synthesis. 

Figure 3. Comparison of the structures of the MnP-1 and UP-H8 genes (26,27). The structures of the genes from the translation initiation codons to the termination codons are shown. Exons are indicated by shading. The putative signal peptides and propeptide are indicated by crosshatching. The positions of the distal His (His 46 and 47), distal Arg (Arg 42 and 43), and proximal His (His 173 and 176) for MnP-1 and LiP-H8, respectively, are also indicated. (Reproduc with permission from Godfr, B. J. Mayfield, M. B. Brown, J. A. Gold, M. H. Gene 1990, in press. Copyright 1990, Elsevier Science Publishers B. V.)...

Then the peptidyl-tRNA at the A site is translocated to the P site by the ribosome moving along the mRNA a codon at a time, exposing the A site for a new aminoacyl-tRNA appropriate for the particular codon, and a repeat of the elongation process occurs. The cycles of elongation and translocation continue until a termination codon is reached, and the peptide or protein is then hydrolysed and released... 

While readthrough is usually a detrimental process, in some cases it can help to suppress problems, e.g. arising from premature stop codons present on the DNA level. This type of readthrough, also termed nonsense suppression, leads to the generation of a fraction of the full length protein in addition to the shortened version. Omnipotent suppressors cause nonsense suppression of all three termination codons. In this process, a near cognate tRNA successfully competes with the termination factors such that amino acid incorporation rather than premature termination of translation occurs. Omnipotent suppression can be caused by mutations in various factors involved in the process of translation termination. Nonsense suppression can also result from an aa-tRNA that decodes a termination codon (suppressor tRNA) in this case only one of the termination codons is efficiently suppressed (Hawthorne and Leupold 1974 Stansfield and Tuite 1994). 

Kushnirov VV, Kryndushkin DS, Boguta M, Smirnov VN, Ter-Avanesyan MD (2000) Chaperones that cure yeast artificial [PSI-h] and their prion-specific effects. Curr Biol 10 1443-1446 Lai C-H, Chun HH, N as SA, Mitui M, Gamo KM, Du L, Gatti RA (2004) Correction of ATM gene function by aminoglycoside-induced read-through of premature termination codons. Proc Natl Acad Sci USA 101 15676-15681... 

Lee SI, Umen JG, Varmus HE (1995) A genetic screen identifies cellular factors involved in retroviral-1 frameshifting. Proc Natl Acad Sci USA 92 6587-6591 Leeds P, Peltz SW, Jacobson A, Culbertson MR (1991) The product of the yeast UPFl gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev 5 2303—... 

The ribosome can carry two aminoacyl-tRNAs simultaneously. In the chain elongation stage, the growing polypeptide is carried on one of these tRNAs. The chain is transferred to the second tRNA, which adds its amino acid to the growing peptide, and displaces the first tRNA. The ribosome then moves one codon along the mRNA to allow the next to be read. Termination of protein synthesis involves the release of the completed polypeptide, expulsion of the last tRNA, and dissociation of the ribosome from the mRNA. This is signaled by specific termination codons (UAA, UAG, or UGA) in the mRNA and requires the participation of various release factors. 

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