DNA Structure and Chromatin

Genome Library analogy (chromosome, gene, and DNA)

Chromosome: book

Gene: Chapter

DNA: text

Central Dogma

DNA → RNA → Protein = gene expression

Because cells in the same body are different, do they have the same genome still?

The chromosomes and genes will still be the same, but the differenitaiton is due to variances in transcription of genes

How many genes are in a chromosome?

Thousands of genes

Definition of genes + what it includes

Sequences of DNA that are transcribed to RNA, and it includes transcribed sequences and sequences that control transcription

Does all DNA get transcribed? What about RNA from translation?

Not all DNA will get transcribed like how now all RNA will be translated to protein

In humans, what percentage of the genome is noncoding repetitive DNA?

~50% (so only a small number of genes actually encodes for proteins)

What are the five important characteristics of genetic material (and explain each one)

1. Replication: must be able to make exact copies of itself to be passed down during cell division

2. Stability-genetic material must be structurally and chemically sound to carry large amounts of complex material

3. Mutability-stable but should have the potential for occasional mutations for genetic variation (genetic material differs between individuals of the same species and outside of the species)

4. Expression: must be read by cell to encode phenotype

5. Heritability: must be passed down from parent to offspring

What does the primary structure of DNA (and RNA) incude

We are looking at the foundational materials needed for DNA/RNA, and the molecule is very long and consists of a chain of many repeating units linked together

What are the repeating units of DNA?

Pentose sugar (base), phosphate group (left), and nitrogenous base (right)

what joins and forms pentose sugar and phosphate groups when combined

The creation of a phosphodiester backone of the polypeptide chains,

What are some difference between RNA snd DNA (3)?

1. RNA is single stranded (DNA is double straded)

2. RNA uses uracil instead of thymine

3. RNA has on more oxygen overall to allow for specific recognition of nucleic acid and make RNA less stable (relly unstable)

What gives DNA and RNA its negative charge? How much more of an impact does it have?

The phosphate group; it helps with functionality + order

What is the phosphate group bonded to?

the 5’ end of carbon atom of the sugar

List nitrogenous bases in DNA and RNA

DNA: adenine, guanine, cytosine and thymine

RNA: adenine, guanine, and cytosine, and uracil

What are purines? pyramidines?

Purines: Guanine and adenine (two rings)

Pyrimidines: cytosine, thymine, and uracil (one ring)

What is the ratio between base pairs?

They are in a 1:1 ratio

C:G is 1:1

A:T is 1:1

So A+G (purines) = T+C (pyramidines)

What are the parts of a nucleotide?

Base+deoxyribose (dN, dG, dA, dT, dC) + phosphate group

What are the similarities and differences in RNA nomenclature?

RNA has the same nomenclature, but when talking about the sugar, it is ribose. So you do G instead of dG, GMP instead of dGMP, and GTP and dGTP

Describe the nature of the polynucleotide strands in DNA (like direction wise)

They are antiparallel (5’ to 3’ and the other is 3’ to 5’)

How are the nucleotides (specifically the backbone) joined together in a single strand?

Phosphodiester bonds occur between 5’PO4 and 3’-OH of each nucleotides

How are complementary bases in DNA bonded together between the complementary strands?

Hydrogen bonds form between the complementary bases (intermolecular=between two molecules)

What is a hydrogen bond?

a weak electrostatic interaction between a hydrogen atom covalently bonded to an electronegative atom (N and O) and another electronegative atom

How many hydrogen bonds form between A-T base pairs? What about C-G base pairs?

A-T have two hydrogen bonds

C-G have three hydrogen bonds

What is the impact on having more C-G bonds in a DNA molecule?

the harder it will be to separate the DNA molecules (so the DNA molecules that need to separate are mostly A-T rich); it also requires more energy to denature G-C base pairs than A-T base pairs

What is another name for complementary base pairing?

Hybridiziation

Denaturation vs renaturation?

Denaturation: double strand to two single strands

Renaturation: 2 single strands to one double strand (also known as reannealing)

In the lab, what can be used to denature DNA

alkali and heat

What is the melting temperature

The temperature at which DNA becomes denatured

Spatially in the DNA molecule, where is the sugar vs the base?

Sugar: on the outside (phosphate backbone)

Base: middle

What interval are nucleotides base pairs spaced along the DNA duplex?

3.4 Å

One helical turn occurs every…

10 bp

What creates major and minor grooves?

Helical turns of the two strands

What is the significance of the major groove?

It is where many proteins bind for cellular processes

Major vs minor groove on DNA strand

Major: looks bigger and longer

Minor: skinnier

What is the significance of intramolecular hydrogen bonding in DNA and RNA?

This is when hydrogen bond occurs within one molecule of DNA or RNA (intermolecular is within two molecules) (within single strand), and it can form structures, such as the hairpin and stem

Describe the shape of hairpin vs stem shape?

Hairpin has look + stem

Stem has just the stem

What does ss DNA and ds DNA mean

ss DNA = single stranded DNA
ds DNA = double stranded DNA

What does each one equal to in terms of bases

1 base pair (bp)

1 kilobase-pair (Kbp) or kilobase (Kb)

1 megabase pair (Mbp) or megabase (Mb)

1 bp=1 pair of H-bonded bases in ds DNA

1 Kb = 1000 bases

1 Mb = 1000 Kb

Does prokaryotic DNA have telomeres?

No, they have circular DNA; telomeres are mainly seen in linear DNA

What are the three levels of DNA structure (describe what each one is)

Primary: nucleotide sequence

secondary: double stranded helix

tertiary: higher holding that takes place in cells

What is supercoiled DNA? What are the two types? Describe their differences (include rotation description)

Additional winding/twisting of double helix DNA

Two types: positive supercoiling and negative supercoiling

Positive: clockwise direction of winding (overrotated)

Negative: counterclockwise (underrotated)

How is most prokaryotic and eukaryotic supercoiled (positive or negative)? Why?

They are negatively supercoiled because it is easier to separate strands for replication and transcription

Function of topoisomerase in coiling?

It adds or removes rotations in DNA by breaking and then rejoining DNA strands

How are eukaryotic chromosomes organized? Why/what is in eukaryotic chromosomes)

As chromatin (because the chromosomes have double stranded DNA helix AND proteins)

Definition of chromatin

DNA and associated proteins of a chromosome

What are the proteins for in chromatin?

For condensation, segregation, and organization of chromosomes

Proportion of DNA and protein in each chromosome

½ DNA and ½ chromsomoes

For the proteins in chromosomes, what kind are they and what are their proportions?

Type: histone and nonhistone proteins

Half are histone and the other half are nonhistone

Describe nonhistone proteins/What do they do?

They are diverse and have a variety of functions in the nucleus (ex: structural, chromosome replication, chromosome segregation, and transcription)

ex: DNA polymerases

What are histones (do not forget the three qualities)

small, positively-charged (remember DNA is negatively charged), highly conserved proteins found in all eukaryotes

So histones are highly conserved. What does this mean

There are little differences between human histones and other species’ histones because they are so crucial and essential in the cell as is

What do histones do?

They bind to and neutralize negatively-charged DNA and make up half of all chromatin protein by weight

How many types of histones are there? What are the types?

There are five types: H1, H2A, H2B, and H3, and H4

What is a nucleosome? (describe composition)

An octamer (8 sub units) of four core histones (two of each of H2A, H2B, H3, and H4)

What are nucleosome remodeling and posttranslational modifications of histones important for?

Gene expression

What is the first level of chromatin organization?

the nucleosome

In purified chromatin from cells, one nucleosome is present every (how many bps of DNA)

Every 200 bp of DNA

About how many bp are in 2 turns around nucleosome core octamer?

145-147 bp

How many bp does histone H1 bind to

20 bp as it binds and leaves the nucleosome

What is Histone H1

It can be between nucleosome cores and also on them by assisting with the wrapping of DNA on the nucleosome

How many bp is the linker DNA between nucleosome cores

30-40 bp

How do we describe nucleosomes on DNA

Like beads on a string

How does chromatin affect gene expression?

If it is supercoiled, no promoter can be exposed (and no essential proteins can bind). Therefore, the gene cannot be expressed (for example via transcription)

What are the different forms chromatin? And what is the difference (2)

Euchromatin vs heterochromatin

Euchromatin has DNA being accessible while heterochromatin has DNA that cannot be expressed

What are the two types of heterochromatin

Constitutive: usually/always transcriptionally inactive; tightly condensed/darkly stained regions of chromosomes; highly compacted even during interphase VS

Facultative: condensed or relaxed under specific conditions (aka can be modified to be accessible/not always inaccessible)

What are four major transformations to molecular genetics? (and describe)

Recombinant DNA technology: where DNA from different sources can be combined

PCR and cloning: DNA fragments can be quickly amplified

DNA sequence identification: DNA sequences can be quickly and accurately read

Genome targeting: genes can be silenced, removed, and/or edited efficiently

What happens with recombinant DNA technology? What is an essential tool?

Set of techniques for locating, isolating, alerting, and studying DNA segments (DNA from other sources can be combined hence the term recombinant) (also known as genetic engineering)

Restriction enzymes help carry this out

What is the function of restriction enzymes?

Make double-strand cuts in specific DNA sequences, so it recognizes specific sequences of bases anywhere within DNA, and the endonuclease will cut phosphodiester bonds of two strands; each enzyme has a unique recognition site

How are restriction fragments generated?

Via digestion of DNA with restriction zymes

What is a recognition site?

usually 4-8 bp of double strand DNA and is where restriction enzymes bind and cut; it is often palindromic (the base sequences are identical when read 5’ to 3’), the recognition site will dictate where the enzyme cuts

Does each enzyme cut in different or the same places?

Each enzyme cuts at the same place based on its specific recognition sequence

What are three examples of recognition sites and type of DNA cleavage? (and describe)

Type of DNA cleavage: 5’ overhang (has sticky ends close to 5’ end or gap is closer to 5’ end), 3’ overhang (sticky ends are on 3’ end and gap is closer to 3’ end), and blunt ends (perfect line/no overhang)

5’ overhang and 3’ overhand are cohesive ends

What is gel electrophoresis?

a mechanism where macromolecules are separated based on size

What are examples of two gels that can be used for gel electrophoresis?

Agarose gel and polyacrylamide gels

Describe the use and function of agarose gel?

Used for restriction mapping, large PCR products, Southern (DNA) blots, and Northern (RNA) blots

Function: separate huge DNA fragments (100-20 kb) (ex: entire chromsome)

Describe the use and function of polyacrylamide gels (PAGE)

Used for: DNA sequencing, resolving small PCR products, separating oligonucleotides, band shifts, and footprints

Function: can be used for native (ds DNA) or denaturing (ss DNA), separates small fragments of 1bp to 1000 bp; separates proteins based on size, conformation, and/or change

What are three factor that affect the migration or final position of a macromolecule after gel electrophoresis? (and describe the trends)

Size: smaller molecules migrate through pores and channels faster (closer to bottom)

Molecular change: negatively charged will migrate faster (going from negative to positive poles in gel)

Molecular shape: tightly condensed globular molecules migrated more quickly than linear

What are three broad steps of gel electrophoresis ?

1. load DNA into wells

2. place gel in buffered aqueous solution, and apply an electrical current

What happens to DNA in gel electrophoresis?

It will be separated based on size. Each sample has its well. and it will migrate through the lane. The positive end is at the bottom, so DNA is migrating towards the bottom. Lastly, migration is dependent on size (smaller travel faster through pores)

How can we see DNA fragments in a gel?

they are stained with a fluorescent dye and the gel is photographed under UV light

How do you determine size of unkown fragments?

Use DNA markers of known size

Fragments at the top will be _____ and the fragments on the bottom will be_____

top=larger

bottom=smaller

How are blotting and gel electrophoresis connected

Nucleic acids or proteins can be transferred from gel to a membrane after electrophoresis

What is the difference between Southern, Northern, and Western blots?

Southern=has transferred DNA

Northern=has transferred RNA

Western=has transferred proteins

What do molecular probes do?

Molecular probes use the transferred molecular substance and identify the specific target molecule on the membrane (helps you find the needle in the haystack or specific macromolecules in a large pool of heterogenous macromolecules)

What are the probes in Southern, Northern, and Western blots?

Southern and Northern blots have DNA or RNA probes that are complementary to target sequences; they can be labeled with radioactivity or linked to an enzyme

Western: the probes are antibodies against target protein (usually linked to an enzyme)

So how many total histones are on one nucleosome

9 (2 of H2A, H2B, H3, and H4 plus one H1)

How many bp per nucleosome

200 bp