Nucleosome assembly is the aggregation, arrangement and bonding together of a nucleosome,

the beadlike structural units of eukaryotic chromatin composed of histones and DNA.
Nucleosome assembly following DNA replication, DNA repair and gene transcription is critical
for the maintenance of genome stability and epigenetic information. Nucleosomes are assembled
by replication-coupled or replication-independent pathways with the aid of histone chaperone
proteins. How these different nucleosome assembly pathways are regulated remains relatively
unclear. Recent studies have provided insight into the mechanisms and the roles of histone
chaperones in regulating nucleosome assembly. A nucleosome is the basic unit of DNA
packaging in eukaryotes, consisting of a segment of DNA wound in sequence around four
histone protein cores. This structure is often compared to thread wrapped around a spool.
Nucleosomes form the fundamental repeating units of eukaryotic chromatin, which is used to
pack the large eukaryotic genomes into the nucleus while still ensuring appropriate access to it
(in mammalian cells approximately 2 m of linear DNA have to be packed into a nucleus of
roughly 10 m diameter). The nucleosome core particle consists of approximately 147 base pairs
of DNA wrapped in 1.67 left-handed superhelical turns around a histone octamer consisting of 2
copies each of the core histones H2A, H2B, H3, and H4; the core consists of a tetramer of H3:H4
complex, and 2 dimer of H2A: AND H2B. Core particles are connected by stretches of "linker
DNA", which can be up to about 80 bp long. Technically, a nucleosome is defined as the core
particle plus one of these linker regions. The core histone proteins contain a characteristic
structural motif termed the "histone fold," which consists of three alpha-helices (1-3) separated
by two loops (L1-2). In solution, the histones form H2A-H2B heterodimers and H3-H4
heterotetramers. Histones dimerise about their long 2 helices in an anti-parallel orientation, and,
in the case of H3 and H4, two such dimers form a 4-helix bundle stabilised by extensive H3-H3

interaction. The H2A/H2B dimer binds onto the H3/H4 tetramer due to interactions between H4
and H2B, which include the formation of a hydrophobic cluster. The histone octamer is formed
by a central H3/H4 tetramer sandwiched between two H2A/H2B dimers. Due to the highly basic
charge of all four core histones, the histone octamer is stable only in the presence of DNA or
very high salt concentrations. The current understanding is that repeating nucleosomes with
intervening "linker" DNA form a 10-nm-fiber, described as "beads on a string", and have a
packing ratio of about five to ten. A chain of nucleosomes can be arranged in a 30 nm fiber, a
compacted structure with a packing ratio of ~50 and whose formation is dependent on the
presence of the H1 histone.

Nucleosome remodeling increases the accessibility of DNA that is associated with nucleosomes.
Nucleosome assembly restricts certain regions of DNA , blocking active sites on the DNA from
regulatory proteins, enzymes and transcription factors needed to initiate and carry out reactions.
Therefore nucleosome dynamics makes it possible nucleosome DNA to be available for essential
reactions. Nucleosome remodeling can take 3 forms sliding of histone octamer along DNA,
complete transfer of octamer to another DNA strand, and remodeling of DNA protein
interactions.

2. Determining nucleosome position in the cell. Review the experiment in Box 7.3 and write
notes (page 180-181).
Nucleosomes were first purified by treating chromosomes with non-specific sequence
micrococcal nuclease. Micrococcal nuclease can cleave protein-free DNA sequences rapidly, but
cleaves protein associated DNA poorly. Limited treatment of chromosomes with this enzyme
yields nuclease-resistant population of DNA that are associated with histones. These DNA
molecules are 160-220 bps in length and are tightly wrapped around the two copies of histones
H2A, H2B, H3, H4. Limited treatment of chromosomes by micrococcal nuclease yields DNA
tightly wrapped around histone core, and at least one unit of linker DNA; whereas a more
extensive treatment yields only DNA histone core that is 147 bp called nucleosome particle core.
To measure the average length of DNA associated with core histone, firstly chromatin is treated
mildly with the micrococcal nuclease. This results in cuts in some of the linker DNA. The DNA
is extracted from the proteins and is subjected to a gel electrophoresis. Electrophoresis reveals a
ladder of fragments that are multiples of the average nucleosome nucleosome distance. Further
digestion would result in the cleavage of the linker DNA and the formation of a nucleosome core
particles and a single fragment 147 bp.
Write a detailed account of nucleosome- remodeling complex, and how it work. Give
illustrated drawings.
Eukaryotic genomes are ubiquitously associated into chromatin; however, cells must spatially
and temporally regulate specific loci independently of bulk chromatin. In order to achieve the
high level of control required to co-ordinate nuclear processes such as DNA replication, repair,
and transcription, cells have developed a variety of means to locally and specifically modulate

chromatin structure and function. One of ways the cell modulates chromatin structure and
function is through ATP dependent nucleosome remodeling. Nucleosome remodeling is
regulated by nucleosome remodeling complexes- multi-protein complexes that facilitate changes
in the nucleosome position, or its interaction with DNA using energy from the hydrolysis of
ATP. There are three common modes of nucleosome remodeling (1) sliding of histone
ocatmer along DNA (2) a complete transfer of the histone ocatmer from one DNA molecule to
another (3) the remodeling of nucleosome to increase access to DNA. Nucleosome movement by
the sliding along the DNA exposes sites for DNA- binding proteins. Remodeling allows the
association of the DNA-binding proteins without alter their position on the DNA.
Go on-line and do journal searches for current information on the human genome
example total size number of genes, repetitive DNA etc.
The human genomethe sum total of hereditary information in a person. The human genome is
found to contain 3 billion base pairs, and approximately 30,000 genes. Completed in 2003, the
Human Genome Project (HGP) was a 13-year project coordinated by the U.S. Department of
Energy and the National Institutes of Health. The Project goals were to

identify all the approximately 20,000-25,000 genes in human DNA,

determine the sequences of the 3 billion chemical base pairs that make up human DNA,

store this information in databases,

improve tools for data analysis,

transfer related technologies to the private sector, and

address the ethical, legal, and social issues (ELSI) that may arise from the project.

According to ScienceMag, issue date September 2012, scientists deciphering the human genome
found, to their surprise, that these protein-coding genes took up less than 3% of the genome. In
between were billions of other bases that seemed to have no purpose. Now a U.S.-funded project,
called the Encyclopedia of DNA Elements (ENCODE), has found that many of these bases do,
nevertheless, play a role in human biology: They help determine when a gene is turned on or off,
for example. As part of ENCODE, 32 institutions did computer analyses, biochemical tests, and
sequencing studies on 147 cell typessix fairly extensivelyto find out what each of the
genome's 3 billion bases does. About 80% of the genome is biochemically active, ENCODE's
442 researchers report today in Nature, weekly science journal. Some of these DNA bases serve
as landing spots for proteins that influence gene activity. Others are converted into strands of
RNA that perform functions themselves, such as gene regulation. (RNA is typically thought of as
the intermediary messenger molecule that helps make proteins, but ENCODE showed that much
of RNA is an end product and is not used to make proteins.) And many bases are simply places
where chemical modifications serve to silence stretches of our chromosomes. ENCODE's results
are changing how scientists think about genes. It found about 76% of the genome's DNA is
transcribed into RNA of one sort or another, way more than researchers had originally expected.
That DNA includes slightly less than 21,000 protein-coding genes (some researchers once
estimated we had more than 100,000 such genes); "genes" for 8800 small RNA molecules and
9600 long noncoding RNA molecules, each of which is at least 200 bases long; and 11,224
stretches of DNA that are classified as pseudogenes, "dead" genes now known to really be active
in some cell types or individuals.
According to PLOS Genetics online journal, research has found that theeEukaryotic genomes
contain millions of copies of transposable elements (TE) and other repetitive sequences. Indeed,

approximately half of the sequence content of typical mammalian genomes tends to be annotated
as TEs and simple repeats by conventional annotation methods. By contrast, only about 510%
of mammalian and vertebrate genome sequences comprise genes and known functional elements

RNA structuregive details of the primary secondary and territory structure and give
enzyme activity (pg. 127-132).
RNA is a polynucleotide strand made up of ribonucleotide phosphates. A ribonucleotide
phosphate consists of a pentose ring with a 2` OH group, a phosphate group bonded to the 5`
carbon, and a nitrogenous base bonded to the 1` carbon. The nitrogenous bases are adenine,
guanine, uracil and cytosine. The primary structure is the sequence of nucleoside
monophosphates (usually written as the sequence of bases they contain). The secondary
structure refers to the shape the RNA assumes as a result of the primary structure. Tertiary
structure refers to large-scale folding in a linear polymer that is at a higher order than secondary
structure. The tertiary structure is the specific three-dimensional shape into which an entire
chain is folded. Unlike DNA, RNA is rarely composed of two strands base paired with each
other. Instead, RNA exists as a single-stranded entity, though extensive regions of many RNAs
may form double helices within themselves by the base pairing rules. The three predominant
forms of RNA are all involved in translating the genetic information in the sequence of bases in
DNA to a sequence of amino acids in proteins. They are called messenger RNA (mRNA),
transfer RNA (tRNA), and ribosomal RNA (rRNA). mRNA is made directly from DNA, so
mRNA carries the genetic information in the DNA sequence from the cell nucleus to the
ribosomes where proteins are made. Information is organized in DNA (and mRNA) in a

sequence of three nucleotides called a codon. One codon specifies the incorporation of a specific
amino acid into a protein. tRNAs translate the genetic code. One end of the tRNA contains a
three nucleotide sequence called the anticodon loop that is complementary to the codon of the
mRNA. The other end of the tRNA is covalently attached to a specific amino acid. Because the
amino acid carried by a tRNA is specific for each anticodon and each anticodon is
complementary to the codons in mRNA, the tRNA provide the link between nucleic acid
sequence and amino acid sequence for a protein during translation. This process, which occurs
on ribosomes, sequentially incorporates amino acids corresponding to the order of codons in the
mRNA. tRNAs contain numerous chemical modifications to the bases within them. Examples
include pseudouridine, ribothymidine, and dihydrouridine rRNA is a component of the
ribosomes where translation (protein synthesis) is occurring. Another type of RNA in eukaryotic
cells, called snRNA (for small nuclear RNA) helps process some RNAs after they are made.
After being capped, the pre-mRNA becomes complexed with a number of small nuclear
ribonucleoprotein particles (snRNPs), which are themselves complexes of small nuclear RNAs
(snRNAs) and special splicing enzymes. The snRNP--pre--mRNA complex is called a
spliceosome. snRNAs recognize and bind intron--exon splice sites by means of complementary
sequences. Some RNA molecules, called ribozymes are capable of catalyzing chemical
reactions. Many natural ribozymes catalyze either the hydrolysis of one of their own
phosphodiester bonds, or the hydrolysis of bonds in other RNAs, but they have also been found
to catalyze the aminotransferase activity of the ribosome.
References:

Reece, Jane; Campbell, Neil (2006). Biology. San Francisco: Benjamin Cummings
Alberts, Bruce (2002). Molecular biology of the cell (4th ed.). New York: Garland
Science. p. 207.

Watson, James D. Tania A. Baker. Stephen P. Bell. Molecular Biology of The

Gene.(2008). San Francisco: Benjamin Cummings
The Human Genome Project Information. U.S. Department of Energy Office of
Science, Office of Biological and Environmental Research. Retrieved on March 13 2013
from http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml