Charge neutralization. Given the histone amino acid sequences illustrated below, estimate the charge of a histone octamer at pH 7. Assume that histidine residues are uncharged at this pH. How does this charge compare with the charge on 150 base pairs of DNA?

Histone H2A

MSGRGKQGGKARAKAKTRSSRAGLQFPVGRVHRLLRKGNYSERVGAGAPVYLAAVLEYLTAEILELAGNA

ARDNKKTRIIPRHLQLAIRNDEELNKLLGRVTIAQGGVLPNIQAVLLPKKTESHHKAKGK

Histone H2B

MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSVYVYKVLKQVHPDTGISSKAMGIMNSFVNDI

FERIAGEASRLAHYNKRSTITSREIQTAVRLLLPGELAKHAVSEGTKAVTKYTSSK

Histone H3

MARTKQTARKSTGGKAPRKQLATKAARKSAPSTGGVKKPHRYRPGTVALREIRRYQKSTELLIRKLPFQR

LVREIAQDFKTDLRFQSAAIGALQEASEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA

Histone H4

MSGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRISGLIYEETRGVLKVFLENVIRDA

VTYTEHAKRKTVTAMDVVYALKRQGRTLYGFGG

Chromatin immunoprecipitation. You have used the technique of chromatin immunoprecipitation to isolate DNA fragments containing a DNA-binding protein of interest. Suppose that you wish to know whether a particular known DNA fragment is present in the isolated mixture. How might you detect its presence?

Around we go. Assuming that 145 base pairs of DNA wrap around the histone octamer 1.75 times, estimate the radius of the histone octamer. Assume 3.4 Å per base pair and simplify the calculation by assuming that the wrapping is in two rather than three dimensions and neglecting the thickness of the DNA.

Nitrogen substitution. Growth of mammalian cells in the presence of 5-azacytidine results in the activation of some normally inactive genes. Propose an explanation.

959

A new domain. A protein domain that recognizes 5-methylcytosine in the context of double-stranded DNA has been characterized. What role might proteins containing such a domain play in regulating gene expression? Where on a double-stranded DNA molecule would you expect such a domain to bind?

Hybrid receptor. Through recombinant DNA methods, a modified steroid hormone receptor was prepared that consists of an estrogen receptor with its ligand-binding domain replaced by the ligand-binding domain from the progesterone receptor. Predict the expected responsiveness of gene expression for cells treated with estrogen or with progesterone.

Different modifications. What is the effect of acetylation of a lysine residue on the charge of a histone protein? Of lysine methylation?

Transformer. The following amino acid sequence of one of the four transcription factors is used to generate iPS cells:

HTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCD

RAFSRSDHLALHMKRHF

This transcription factor belongs to one of the three structural classes discussed in Section 32.2. Identify the class.

Coverage. What percentage of the DNA sites in yeast are accessible, assuming that the fraction of sites observed for GAL4 is typical? To how many base pairs of the 12-Mb yeast genome does this percentage correspond?

Count the methyl groups. Examination of the histone modifications of a gene reveals an abundance of histone H3 with lysine 27 modified with a single methyl group. Does this suggest that this gene is activated or repressed? How would your answer change if many lysine 27 residues were modified with three methyl groups each?

Iron regulation. What effect would you expect from the addition of an IRE to the 5′ end of a gene that is not normally regulated by iron levels? To the 3′ end?

Predicting microRNA regulation. Suppose that you have identified an miRNA that has the sequence 5′-GCCUAGCCUUAGCAUUGAUUGG-3′. Propose a strategy for identifying mRNA that might be regulated by this miRNA, given the sequences of all mRNAs encoded by the human genome.

Acetyltransferases. Propose a mechanism for the transfer of an acetyl group from acetyl CoA to the amino group of lysine.

Limited restriction. The restriction enzyme HpaII is a powerful tool for analyzing DNA methylation. This enzyme cleaves sites of the form 5′-CCGG-3′ but will not cleave such sites if the DNA is methylated on any of the cytosine residues. Genomic DNA from different organisms is treated with HpaII and the results are analyzed by gel electrophoresis (see the adjoining patterns). Provide an explanation for the observed patterns.