Question

Genetics students were studying protein synthesis in the protozoan Tetrahymena. The students isolated a portion of the nucleic acid sequence, which codes for the C terminus of a Tetrahymena protein, which is:

AUU AUG UAU AAG UAG GUC GCA UAA ACA CAA UUA UGA GAC UUA

   I        M      Y       K        Q     V      A      Q       T       Q        L    stop

However, the students were unable to translate purified Tetrahymena mRNA in an in vitro system as the translation products were mostly small polypeptides. The brilliant students decided to carry out several control experiments. First, they translated pure mRNA from tobacco mosaic virus (TMV) which encoded a 116 kd (kilodalton) protein. TMV mRNA translated in vitro giving a major band at 116 kd which is the expected product with a minor band about 50 kd larger. When Tetrahymena RNA is added, there is a significant increase in the higher molecular weight product. When the genetics students added Tetrahymena cytoplasm (minus the ribosomes), the TMV mRNA translated into the higher molecular weight product and the previously inactive Tetrahymena mRNA was also translated into high molecular weight proteins which was confirmed by leaving out the TMV mRNA.                                    

1. What is unusual about the sequence data for the Tetrahymena protein? (6 points)

2. What were the two Tetrahymena cytoplasmic components required for the efficient in vitro translation of Tetrahymena RNA? (4 points)

Answer 1

1. The Tetrahymena has the usual termination codon UAA and UAG coding for amino acid glutamine. As per Wobble hypothesis, UAA codon can have 3'AUU5' or 3'AUI 5’ anticodons. These anticodons recognize UAA/G and UAC/A/U respectively as per Wobble hypothesis. The two tyrosine codons in Tetrahymena are coded by UAU and UAC. Hence, the 3’ AUU5’ codon will bind to 5’UAA3’ and 5’ UAG3’. In humans, glutamine is coded by 5’CAA3’, 5’CAG3’.

The stop codon now in Tetrahymena is UGA only. AUU is also codon coding for inosine. Inosine is a base normally seen in anticodons. In this Tetrahymena, AUU is coding for inosine.

2. Tetrahymena cytoplasm has a tRNA gluUmUA which recognizes the codon UAA and UAG. This tRNA has an unmodified uridine in place of ribosylthymine at the position 54. Further, there is a tRNAGlnCUA , which is known to recognize UGA as the termination codon. Normally tRNA CUA codes for leucine. There is another tRNAglnUmUG, which is known to suppress UAA.

These specific tRNAs are known as isoacceptor tRNAs. The normal tRNAGlnUmUG is also present. Their role is to use UAG and UAA as codons for glutamine and only use UGA as termination codon. Thus, when the cytoplasm of Tetrahymena is added, UAG and UAA are not used as stop codons and hence, the protein formed will be longer as will the TMV protein.