Take-away Exam I

Answer any four of the five following questions.

PDFs of cited manuscripts will be emailed.

Question1: Hypothetical data analysis.

A. In class two models regarding the mechanism of C protein action were discussed.

Model 1. C protein operates independently of other viral proteins.

To possibly distinguish between these two models a cross-complementation assay was performed. Discuss whether the following two different sets of hypothetical results are consistent with one or both of the models.

B. The following hypothetical cross-inhibition and cross-complementation yielded the following results. Formulate a model that is consistent with the data.

Question 2: Protein Homologies

 The amino acid sequences of the three C proteins are given below. There are several online programs that will perform pair wise alignments of proteins. For example, LALIGN (http://www.ch.embnet.org/software/LALIGN_form.html). If you use LALIGN, identical amino acids are depicted with a “:” whereas similar amino acids are depicted with a “.”

 øX174 C protein: MRKFDLSLRSSRSSYFATFRHQLTILSKTDALDEEKWLNMLGTFVKDWFRYESHFVHGRDSLVDILKERGLLSESDAVQPLIGKKS

 G4 C protein:       MRKFNLNLKNSRSSYFATFRHHLNVLAKTDALDEEKYLNMLGALLKDWFRYEEHFVHGKQSMLDILKERGLLSTSSTDTNHKGN

 alpha3 C protein: MLSSTRYYASYRATLTKQLMFLTKADFTNDDEKWLNAFGNLLRQWFQIEDWKGNHKKLLDDLKKRDYL

 1. The behavior of chimeric proteins or proteins containing deletions can be very valuable when investigating the function of a protein domain. As the name suggests, chimeric proteins contain are made by combining two different wild-type sequences. Examples of how chimeric proteins can be used in research can be found in  Uchiyama et al. (2007) and Burch and Fane (2000). Design a chimeric C protein and describe how it may behave in various complementation and inhibition assays.

 2. When writing this question I made the following observation. In the alpha3 genome, there is an intercistronic region between the end of gene A and the beginning of gene C. In other words, there are untranslated nucleotides between the two genes. By contrast, in the øX174 genome, there is no intercistronic region. The stop codon of gene A overlaps with the start codon of gene C.

What could be learned, or perhaps already learned about viral evolution and C protein function by just introducing the following mutations into the alpha3 genome.

Wild-type: GGCGGTAAGTGACCCGGATTTTTCTCAGGAGAAAATTCATGCTGAGCTC

Mutant:    GGCGGTAAGTGACCCGGAGTTTTTCTATGAGAAAATTCGACCTGAGCTC

In class, we discussed the Luria and Delbruck slot machine experiment and how it relates to the isolation of independent mutations. In Gordon et al (In press), the research involved a large number of initial mutations. Isolating independent suppressors of so many initial mutations would have been extremely laborious, time consuming, and expensive. Instead a cross-suppressor analysis was conducted. It is highly likely that this alternative approach was just as effective. Why?

Question 4: Informational suppression.

A. The amber codon is UAG. Which tRNA molecules could theoretically be mutated to become informational suppressors via a single base substitution. (Hint: consult a genetic code).

B. Although many informational suppressors are theoretically possible, only three amber suppressors; a mutant glutamine tRNA, a mutant serine tRNA, and a mutant tyrosine tRNA; are tolerated. Explain why other mutant tRNA’s may be lethal.

C. There are three stops amber (UAG), ochre (UAA) and opal (UGA) found at the end of genes. In general, informational amber suppressors have less adverse side effects on cellular physiology than ochre and opal informational suppressors. How may this relate to stop codon usage in E. coli?

 D. Host cell genes usually contain two adjacent stop codons as opposed to one. Why may this be the case?

Question 5: C protein structure and function

In Burch and Fane (2000), a mutation that conferred resistance to a foreign external scaffolding protein (see Figure 4). Although the focus of that research project was completely unrelated to our studies, that mutation (called forD^R) relates to the C protein project. Based on the C protein sequences in Question 2 and the results of your homology comparisons, formulate a hypothesis and an experiment to test it.