Question

You have identified a gene that produces a small RNA. You find that it is transcribed by RNA polymerase III. Because it enodes an RNA that is not related to tRNAs or the 5S ribosomal RNA you wonder if the same factors are required for transcription of your gene. You find that TFIIIB, TFIIIC and RNAPIII are not sufficient to transcribe your gene in vitro. You decide to try to purify the factors that are needed for transcription of your gene. You start with a cell extract that together with RNAPIII transcribes your gene in vitro as indicated in the figure below. You fractionate your extract into three fraction, W, X and Y and assay combinations of those fractions for the ability to support transcription in vitro. You obtain the results shown in the image below. Answer the following 2 parts (i,ii) based on this information.

Cell or nuclear Fractionate extract W Х Y Txn Reaction 1. Extract only 2. RNAP only 3. Ext. + RNAP 4. RNAP+X+Y+Z 5. RNAP+X+Y 6. RNAP+X+Z 7. RNAP+Y+Z | +++

i. What is the minimal set of components required for transcription of your gene in vitro?

A. RNAPIII

B. RNAPIII + X = Y + Z

C. X + Y + Z

D. RNAPIII + X

E. RNAPIII + Y

F. RNAPIII + Z

G. RNAPIII + X + Y

H. RNAPIII + X + Z

I. RNAPIII + Y + Z

ii. Assuming your initial round of fractionation yielded only partially purified fraction, which fractions would you need to continue to fractionate to identify the proteins involved in transcription of your gene? Choose the single best answer.

A. X

B. Y

C. Z

D. X + Y

E. X + Z

F. Y + Z

G. X + Y + Z

Answer 1

I) Basal transcription factors along with RNAPIII aren't sufficient for transcription of the gene. Only the cell extract can't cause transcription as without RNAPIII transcription of the gene won't occur. Only RNAPIII can't transcribe the gene as the transcription process requires additional factors for expression of the gene. From the results of the combination assay we can clearly see that RNAPIII along with the fractionates X and Y can express the gene in vitro but not with the combinations (X+Z) or (Y+Z).

So the minimal set of components required for in vitro transcription of the gene is set G) RNAPIII+X+Y

II) From the results of the combination assay it is quite evident that RNAPIII requires two other factors for the transcription of the gene in vitro. One factor might be in the fraction X and the other factor might be in fraction Y. So we need to continue fractionating fractions X+Y to get the proteins required for the transcription of our gene.