DNA footprint protection is a method that determines whether proteins bind to a specific sample of DNA and thus protect part of the DNA from random enzymatic cleavage by DNase I. A 400-bp segment of cloned DNA is thought to contain a promoter. The cloned DNA is analyzed by DNA footprinting to help determine if it has the capacity to act as a promoter sequence. The accompanying gel has two lanes, each containing the cloned 400-bp DNA fragment treated with DNase I to randomly cleave unprotected DNA. Lane 1 is cloned DNA that was mixed with RNA polymerase II and several TFII transcription factors before exposure to DNase I. Lane 2 contains cloned DNA that was exposed only to DNase I. RNA pol II and TFIIs were not mixed with that DNA before adding DNase I. What additional genetic experiments would you suggest to verify that this region of cloned DNA contains a functional promoter?

DNA footprint protection is a method that determines whether proteins bind to a specific sample of DNA and thus protect part of the DNA from random enzymatic cleavage by DNase I. A 400-bp segment of cloned DNA is thought to contain a promoter. The cloned DNA is analyzed by DNA footprinting to help determine if it has the capacity to act as a promoter sequence. The accompanying gel has two lanes, each containing the cloned 400-bp DNA fragment treated with DNase I to randomly cleave unprotected DNA. Lane 1 is cloned DNA that was mixed with RNA polymerase II and several TFII transcription factors before exposure to DNase I. Lane 2 contains cloned DNA that was exposed only to DNase I. RNA pol II and TFIIs were not mixed with that DNA before adding DNase I. Approximately what length is the DNA region protected by RNA pol II and TFIIs?

Assume that a mutation affects the gene for each of the following eukaryotic RNA polymerases. Match each mutation with the possible effects from the list provided. More than one effect is possible for each mutation.

Some pre-mRNA is not synthesized.

Suppose you have a 1-kb segment of cloned DNA that is suspected to contain a eukaryotic promoter, including a TATA box, a CAAT box, and an upstream GC-rich sequence. The clone also contains a gene whose transcript is readily detectable. Your laboratory supervisor asks you to outline an experiment that will (1) determine if eukaryotic transcription factors (TF) bind to the fragment and, if so, (2) identify where on the fragment the transcription factors bind. All necessary reagents, equipment, and experimental know-how are available in the laboratory. Your assignment is to propose techniques to be used to address the two items your supervisor has listed and to describe the kind of results that would indicate binding of TF to the DNA and the location of the binding.

Assume that a mutation affects the gene for each of the following eukaryotic RNA polymerases. Match each mutation with the possible effects from the list provided. More than one effect is possible for each mutation.

Pre-mRNA does not have introns removed.

DNA footprint protection is a method that determines whether proteins bind to a specific sample of DNA and thus protect part of the DNA from random enzymatic cleavage by DNase I. A 400-bp segment of cloned DNA is thought to contain a promoter. The cloned DNA is analyzed by DNA footprinting to help determine if it has the capacity to act as a promoter sequence. The accompanying gel has two lanes, each containing the cloned 400-bp DNA fragment treated with DNase I to randomly cleave unprotected DNA. Lane 1 is cloned DNA that was mixed with RNA polymerase II and several TFII transcription factors before exposure to DNase I. Lane 2 contains cloned DNA that was exposed only to DNase I. RNA pol II and TFIIs were not mixed with that DNA before adding DNase I. Explain why this gel provides evidence that the cloned DNA may act as a promoter sequence.

The accompanying illustration shows a portion of a gene undergoing transcription. The template and coding strands for the gene are labeled, and a segment of DNA sequence is given.

For this gene segment identify the direction in which the promoter [TIP 2] for this gene is located.