Tryptophan codons

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).

UGA is used as a tryptophan codon (instead of a stop codon). 

The leader sequence has an AUG codon that is in-phase with a UGA stop codon together these start-stop signals encode a polypeptide of 14 amino acids. The leader sequence has an interesting feature—at positions 10 and 11 are two adjacent tryptophan codons. 

Premature termination of mRNA synthesis is mediated through translation of the leader peptide. The two tryptophan codons make translation of the leader polypeptide... 

NAtrp levels in the cell are low because tryptophan is limiting, then ribosomes pause at a pair of tryptophan codons in the RNA. This transient ribosomal pausing allows time for an alternate hairpin structure to form in the nascent RNA which disrupts the transcriptional termination signal and RNA polymerase is able to continue down the DNA template and complete transcription (Fig. 28.11). 

If tryptophan is limiting, the ribosome stalls out over the tryptophan codons on the mRNA of the leader sequence. This leaves the mRNA free to form the... 

See Figure 11.14. When the level of tryptophan is low, the trptRNA becomes limiting. This stalls the ribosome over the tryptophan codons on the mRNA. By stalling the ribosome there, the antitermination loop can form, transcription is not aborted, and the full mRNA is produced. If the ribosome does not stall there, the termination loop forms, and the leader mRNA dissociates. 

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 code is degenerate. More than one codon can specify a single amino acid. All amino adds, except Met and tryptophan (Trp), have more than one codon. 

The codons are written in the 5 —>3 direction. The third base of each codon (in bold type) plays a lesser role in specifying an amino acid than the first two. The three termination codons are shaded in pink, the initiation codon AUG in green. All the amino acids except methionine and tryptophan have more than one codon. In most cases, codons that specify the same amino acid differ only at the third base. 

A striking feature of the genetic code is that an amino acid may be specified by more than one codon, so the code is described as degenerate. This does not suggest that the code is flawed although an amino acid may have two or more codons, each codon specifies only one amino acid. The degeneracy of the code is not uniform. Whereas methionine and tryptophan have single codons, for example, three amino acids (Leu, Ser, Arg) have six codons, five amino acids have four, isoleucine has three, and nine amino acids have two (Table 27-3). 

When tryptophan levels are low, the ribosome pauses at the Trp codons in sequence 1. Formation of the paired structure between sequences 2 and 3 prevents attenuation, because sequence 3 is no longer available to form the attenuator structure with sequence 4. The 2 3 structure, unlike the 3 4 attenuator, does not prevent transcription. 

When tryptophan concentrations are high, concentrations of charged tryptophan tRNA (Trp-tRNATrp) are also high. This allows translation to proceed rapidly past the two Trp codons of sequence 1 and into sequence 2, before sequence 3 is synthesized by RNA polymerase. In this situation, sequence 2 is covered by the ribosome and unavailable for pairing to sequence 3 when sequence 3 is synthesized the attenuator structure (sequences 3 and 4) forms and transcription halts (Fig. 28-21b, top). When tryptophan concentrations are low, however, the ribosome stalls at the two Trp codons in sequence 1, because charged tRNATrp is less available. Sequence 2 remains free while sequence 3 is synthesized, allowing these two sequences to base-pair and permitting transcription to proceed (Fig. 28-21b, bottom). In this way, the proportion of transcripts that are attenuated declines as tryptophan concentration declines. 

Model for attenuation in the trp operon, showing ribosome and leader RNA. (a) Where no translation occurs, as when the leader AUG codon is replaced by an AUA codon, stem-and-loop 3.4 is intact, and termination in the leader is favored. (b) Cells are selectively starved for tryptophan so that the ribosome stops prematurely at the tandem trp codons. Under these conditions, stem-and-loop 2.3 can form, and this is believed to lead to the disruption of stem-and-loop 3.4. (c) All amino acids, including excess tryptophan, are present so that stem-and-loop 3.4 is present. 

Fig. 2. Attenuation of the trp operon. (a) When tryptophan is plentiful, sequences 3 and 4 base-pair to form a 3 4 structure that stops transcription (b) when tryptophan is in short supply, the ribosome stalls at the trp codons in sequence 1, leaving sequence 2 available to interact with sequence 3. Thus a 3 4 transcription terminator structure cannot form and transcription continues.

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),... 

The genetic code is not universal but is the same in most organisms. Exceptions are found in mitochondrial genomes where some codons specify different amino acids to that normally encoded by nuclear genes. In mitochondria, the UGA codon does not specify termination of translation but instead encodes for tryptophan. Similarly, in certain protozoa UAA and UAG encode glutamic acid instead of acting as termination codons. 

---