Question

The EU 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 EU students were unable to translate purified Tetrahymena  mRNA in an in vitro  system as the translation products were mostly small polypeptides. The brilliant EU 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 EU 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 protein has unusual stop codons - UAA and UAG. They code for glutamine, and the Wobble hypothesis states that the anticodons for UAA can either be AUU or AUI that recognize UAA/G and UAC/A/U. The codons for tyrosine in the given protein are coded by UAC and UAU which means that the AUU codon will bind to the UAA and UAG codons. It leaves only one stop codon (UGA). Also, in the given protein the AUU is coding of inosine which is normally seen in the anticodons.

2. The presence of the tRNA gluUmUA in the cytoplasm of the tetrahymena helps by recognizing the UAA and UAG codons. The recognition of the termination codon UGA is done by tRNA GlnCUA. Another tRNA supresses the recognition of UAA. These different tRNAs in the cytoplasm of the Tetrahymena allows for a longer formation of the protein because the UAG and UAA are not recognized as the stop codons.