The role of a chaperone in protein synthesis

Authors

  • József Szeberényi

    Corresponding author
    1. Department of Medical Biology, School of Medicine, University of Pécs, H-7624 Pécs, Szigeti 12, Hungary
    • Department of Medical Biology, School of Medicine, University of Pécs, H-7624 Pécs, Szigeti 12, Hungary
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Abstract

Terms to be familiar with before you start to solve the test: chaperones, sucrose density gradient centrifugation, SDS-polyacrylamide gel electrophoresis, Western blotting, translation, ribosomes, polysomes.

Molecular chaperones are protein molecules that play an important role in the folding and membrane translocation of newly synthesized proteins as well as in the assembly of protein complexes. Hsp70 heat shock proteins are the best characterized family of chaperones. The experiments described in this test were performed to study the role of a cytoplasmic yeast Hsp70 (designated Ssb1) in the translation machinery.

A crude cytoplasmic extract was prepared from yeast cells and subjected to sucrose density gradient centrifugation (Fig. 1A). UV absorption at 260 nm was monitored in the gradient (A260).

FIVE-CHOICE COMPLETION

Select the one best answer.

  • ____ Which of the following components contributed most to the UV absorption curve of Fig. 1A?

    • DNA

    • rRNA

    • mRNA

    • tRNA

    • Ribosomal proteins

  • ____ The materials in peaks I and II in Fig. 1A were successfully separated from each other because

    • the protein/RNA ratio in peak I is higher than in peak II

    • the protein/RNA ratio in peak I is lower than in peak II

    • they have different buoyant densities

    • peak I particles are larger than peak II particles

    • peak I particles are smaller than peak II particles

  • ____ What is the number of ribosomes in the complexes of peak I?

    • 0

    • 1

    • 2

    • 3

    • Cannot be determined

Aliquots of the gradient fractions were subjected to electrophoresis in an SDS-polyacrylamide gel, and Western blots were performed using antibodies raised against the Ssb1 protein or the large ribosomal subunit protein L16. To obtain Fig. 1B, the extract was exposed to mild RNase treatment before centrifugation. Fig. 1C shows the results of a similar experiment; in this case the extract was prepared from sodium azide-treated cells. (Sodium azide allows translating ribosomes to run off the mRNA but prevents reinitiation.) What can be the difference between the ribosomes present in samples B and C?

FOUR-CHOICE ASSOCIATION

In this type of question a set of lettered headings is followed by a list of numbered words or phrases. Select

  • A if the word or phrase is associated with A only;

  • B if the word or phrase is associated with B only;

  • C if the word or phrase is associated with A and B;

  • D if the word or phrase is associated with neither A nor B

  • Ribosomes in the RNase-treated extract

  • Ribosomes from sodium azide-treated cells

  • Both of them

  • Neither of them

  • 4. ____ mRNA sequences are attached to them.

  • 5. ____ Consist of a small and a large subunit.

  • 6. ____ Are mostly present in a polysomal form.

  • 7. ____ Contain the L16 protein.

  • 8. ____ tRNA molecules are attached to them.

  • 9. ____ May carry nascent polypeptide chains.

  • 10. ____ Ssb1 protein is bound to them.

In the second part of the experiment the effect of puromycin treatment was studied on the binding of Ssb1 protein to the polysomes. (Puromycin is an aminoacyl-tRNA analog that is incorporated into the C terminus of the growing polypeptide chain, stops its synthesis, and causes the release of the nascent chain from the ribosome.) Fig. 2 shows the results of an experiment in which yeast extracts were either left untreated (Fig. 2A) or treated with puromycin (Fig. 2B) and then analyzed as described for Fig. 1. Compare the two charts, and solve the following multiple-choice questions.

FOUR-CHOICE ASSOCIATION

In this type of question a set of lettered headings is followed by a list of numbered words or phrases. Select

  • A if the word or phrase is associated with A only;

  • B if the word or phrase is associated with B only;

  • C if the word or phrase is associated with A and B;

  • D if the word or phrase is associated with neither A nor B.

  • Untreated extract

  • Puromycin-treated extract

  • Both of them

  • Neither of them

  • 11. ____ Contains mRNA.

  • 12. ____ Contains polyribosomes.

  • 13. ____ Ssb1 protein binds to its ribosomes.

FIVE-CHOICE COMPLETION

Select the one best answer.

  • 14. ____ Based on the evidence presented in this article, determine the most likely binding site of Ssb1 during protein synthesis:

    • The peptidyltransferase enzyme

    • The small ribosomal subunit

    • The nascent polypeptide

    • mRNA sequences bound to the ribosomes

    • mRNA sequences between ribosomes

CORRECT ANSWERS

  • B

  • E

  • D

  • A

  • C

  • D

  • C

  • A

  • A

  • A

  • C

  • C

  • A

  • C

EXPLANATIONS

Sucrose density gradient centrifugation was used in this experiment to fractionate ribosomes/polysomes by size (MCQ11 2: E; MCQ 3: D); the presence of ribosomes in gradient fractions was monitored by UV absorption of their rRNAs (MCQ 1: B) or by their protein content (MCQ 7: C). Mild RNase treatment digests mRNA sequences between ribosomes in the polysomes (MCQ 6: D) but does not disrupt translational complexes assembled on individual ribosomes (MCQ 4: A; MCQ 5: C; MCQ 8: A; MCQ 9: A). In contrast, treatment with sodium azide or puromycin, as described in the text, leads to premature release of newly synthesized polypeptides from the ribosomes (otherwise puromycin does not affect the polysomal structure; MCQ 11: C; MCQ 12: C). Based on the Western blotting patterns the chaperone Ssb1 specifically binds to the newly synthesized protein molecules (MCQ 10: A; MCQ 13: A; MCQ 14: C).

The test was based on R. J. Nelson, T. Ziegelhoffer, C. Nicolet, M. Werner-Washburne, E. A. Craig (1992) The translation machinery and 70 kd heat shock protein cooperate in protein synthesis, Cell71, 97–105.

Figure FIGURE 1..

Analysis of yeast cytoplasmic extracts by sucrose density gradient centrifugation (upper panels) followed by Western blotting (lower panels). For details see the text.

Figure FIGURE 2..

The effect of puromycin treatment on yeast polysomes. For details see the text.

Footnotes

  1. 1

    The abbreviation used is: MCQ, multiple choice question.

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