MRNA tryptophan

Siuciak, JA, Clark, MS, Rind, HB, Whittemore, SR and Russo, AF (1998) BDNF induction of tryptophan hydroxylase mRNA levels in the rat brain. J. Neurosci. Res. 52 149-158. Sprague, JE, Everman, SL and Nichols, DE (1998) An integrated hypothesis for the serotonergic axonal loss induced by 3,4-methylenedioxymethamphetamine. Neuro toxicology 19 427-A42. Stock, MJ (1997) Sibutramine a review of the pharmacology of a novel anti-obesity agent. Int. J. Obesity 21 (Suppl 1) S25-S29. 

Champier, J., Claustrat, B., Besancon, R. el al. (1997). Evidence for tryptophan hydroxylase and hydroxy-indol-O-methyl-transferase mRNAs in human blood platelets. Life Sci 60, 2191-7. 

The information contained in the DNA (i.e., the order of the nucleotides) is first transcribed into RNA. The messenger RNA thus formed interacts with the amino-acid-charged tRNA molecules at specific cell organelles, the ribosomes. The loading of the tRNA with the necessary amino acids is carried out with the help of aminoacyl-tRNA synthetases (see Sect. 5.3.2). Each separate amino acid has its own tRNA species, i.e., there must be at least 20 different tRNA molecules in the cells. The tRNAs contain a nucleotide triplet (the anticodon), which interacts with the codon of the mRNA in a Watson-Crick manner. It is clear from the genetic code that the different amino acids have different numbers of codons thus, serine, leucine and arginine each have 6 codewords, while methionine and tryptophan are defined by only one single nucleotide triplet. 

The initial hydroxylation of tryptophan, rather than the decarboxylation of 5-HTP, appears to be the rate-limiting step in serotonin synthesis. Therefore, the inhibition of this reaction results in a marked depletion of the content of 5-HT in brain. The enzyme inhibitor most widely used in experiments is parachlorophenylalanine (PCPA). In vivo, PCPA irreversibly inhibits tryptophan hydroxylase, presumably by incorporating itself into the enzyme to produce an inactive protein. This results in a long-lasting reduction of 5-HT levels. Recovery of enzyme activity, and 5-HT biosynthesis, requires the synthesis of new enzyme. Marked increases in mRNA for tryptophan hydroxylase are found in the raphe nuclei 1-3 days after administration of PCPA [6]. 

Cortes, R., Mengod, G., Celada, P. and Artigas, F. p-Chlorophenylalanine increases tryptophan-5-hydroxy-lase mRNA levels in the rat dorsal raphe a time course study using in situ hybridization. J. Neurochem. 60 61-764,1993. 

Bendotti, C., Servadio, A., Forloni, G., Angeretti, N. and Samanin, R. Increased tryptophan hydroxylase mRNA in raphe serotonergic neurons spared by 5,7-dihydroxytrypta-mine. Mol. Brain Res. 8 342-248,1990. 

Garcia-Osta, A., Del Rio, J. and Frechilla, D. Increased CRE-binding activity and tryptophan hydroxylase mRNA expression induced by 3,4-methylenedioxymethamphetamine (MDMA, ecstasy ) in the rat frontal cortex but not in the hippocampus. Mol Brain Res. 126 181-187,2004. 

Transcription of the following sequence of the tryptophan operon occurs in the direction indicated by the arrow. What would be the base sequence of the mRNA produced ... 

FIGURE 28-19 The trp operon. This operon is regulated by two mechanisms when tryptophan levels are high, (1) the repressor (upper left) binds to its operator and (2) transcription of trp mRNA is attenuated (see Fig. 28-21). The biosynthesis of tryptophan by the enzymes encoded in the trp operon is diagrammed at the bottom... 

Regulatory sequence 1 is crucial for a tryptophan-sensitive mechanism that determines whether sequence 3 pairs with sequence 2 (allowing transcription to continue) or with sequence 4 (attenuating transcription). Formation of the attenuator stem-and-loop structure depends on events that occur during translation of regulatory sequence 1, which encodes a leader peptide (so called because it is encoded by the leader region of the mRNA) of 14 amino acids, two of which are Trp residues. The leader peptide has no other known cellular function its synthesis is simply an operon regulatory device. 

Effect of mRNA and Protein Stability on Regulation E. coli cells are growing in a medium with glucose as the sole carbon source. Tryptophan is suddenly added. The cells continue to grow, and divide every 30 min. Describe (qualitatively) how the amount of tryptophan synthase activity in the cells changes with time under the following conditions ... 

As any elementary textbook on molecular biology will relate, the sequences of proteins are stored in DNA in the form of a triplet code. Each amino acid is encoded by one or more triplet combinations of the four bases A, T, G, and C. For example, tryptophan is coded by the sequence TGG. The sequence of triplets is converted into a protein by a process in which DNA is first transcribed into mRNA. This message is then translated into protein on the ribosomes in conjunction with tRNA and the aminoacyl-tRNA synthetases. In prokaryotes, there is a one-to-one relationship between the sequence of triplets in the DNA. and the sequence of amino acids in the protein. In eukaryotes, the DNA often contains stretches of intervening sequences or introns which are excised from the mRNA after transcription (Chapter 1). 

The function of the leader sequence is to fine tune expression of the trp operon based on the availability of tryptophan inside the cell. It does this as follows. The leader sequence contains four regions (Fig. 2, numbered 1-4) that can form a variety of base-paired stem-loop ( hairpin ) secondary structures. Now consider the two extreme situations the presence or absence of tryptophan. Attenuation depends on the fact that, in bacteria, ribosomes attach to mRNA as it is being synthesized and so translation starts even before transcription of the whole mRNA is complete. When tryptophan is abundant (Fig. 2a), ribosomes bind to the trp polycistronic mRNA that is being transcribed and begin to translate the leader sequence. Now, the two trp codons for the leader peptide lie within sequence 1, and the translational Stop codon (see Topic HI) lies between sequence 1 and 2. During translation, the ribosomes follow very closely behind the RNA polymerase and synthesize the leader peptide, with translation stopping eventually between sequences 1 and 2. At this point, the position of the ribosome prevents sequence 2 from interacting with sequence 3. Instead sequence 3 base-pairs with sequence 4 to form a 3 4 stem loop which acts as a transcription terminator. Therefore, when tryptophan is present, further transcription of the trp operon is prevented. If, however, tryptophan is in short supply (Fig. 2b), the ribosome will pause at the two trp codons contained within sequence 1. This leaves sequence 2 free to base pair with sequence 3 to form a 2 3 structure (also called the anti-terminator),... 

We noted earlier the coordinate repression of the enzymes for tryptophan synthesis in the presence of tryptophan. When the level of tryptophan is depleted or reduced, trp mRNA is synthesized in 3-4 minutes and rapidly degraded it has a half-life of some 3 minutes. This allows the bacteria to respond quickly to changing requirements for tryptophan. 

Figure 9.6. Cation-71 interaction in the binding of acetylcholine to the nicotinic acetylcholine receptor, a Incorporation of flu-orinated tryptophan residues into NAR a chains in vivo. Individual tryptophan codons were replaced by amber stop codons (UAA). The mRNA was obtained by transcription in vitro and injected into Xenopus laevis (frog) oocytes, along with a suppressor tRNA that had been acylated with the synthetic fluorinated trp. b Oocytes expressing the modified NAR were analyzed by patch clamp. Currents are plotted as functions of acetylcholine concentration. The EC q increases monotonously with the extent of fluorination. c Replot of the EC q as a function of a derived parameter that describes the strength of the cation-71 interaction (the derivation is beyond me).

The codons of individual tryptophan residues in the cloned a chain were exchanged for an amber stop codon (TAA) and the mutant mRNA was obtained by in vitro transcription. 

Induction of Tryptophan Dioxygenase by Glucocorticoid Hormones The de novo synthesis of tryptophan dioxygenase is induced by glucocorticoid hormones (cortisol in human beings and corticosterone in the rat). This is true induction of new mRNA and protein synthesis indeed. 

Figure 5.26. Complementarity between mRNA and DNA. The base sequence of mRNA (red) is the complement of that of the DNA template strand (blue). The sequence shown here is from the tryptophan operon, a segment of DNA containing the genes for five enzymes that catalyze the synthesis of tryptophan. The other strand of DNA (black) is called the coding strand because it has the same sequence as the RNA transcript except for thymine (T) in place of uracil (U).

A novel mechanism for regulating transcription in bacteria was discovered by Charles Yanofsky and his colleagues as a result of their studies of the tryptophan operon. The 7-kb mRNA transcript from this operon encodes five enzymes that convert chorismate into tryptophan (Section 24.2.10). The mode of regulation of this operon is called attenuation, and it... 

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