DNA Structure and Organization Flashcards
Nucleoid
A region in the bacterial cell where all nucleic acid is contained. Essentially a compact, amorphous mass of DNA located in the center of the cell. It is not membrane enclosed. DNA is stored, replicated, and transcribed to RNA in the nucleoid. There is one chromosome per nucleoid and one nucleoid per bacterial cell
Bacterial DNA
Bacteria has one chromosome that is stored, replicated, and transcribed to RNA in the nucleoid. The DNA is very compact and amorphous, and is located in the center of the cell
Bacterial RNA
Transcribed from DNA to RNA in the nucleoid. It is translated to protein by ribosomes
Bacterial ribosomes
Located outside the nucleoid region. Ribosomes are essentially the only organelles bacteria has
How is a eukaryotic nucleus different from a prokaryotic nucleus?
A eukaryotic nucleus is membrane enclosed
Structure of the eukaryotic nucleus
Has 2 membranes- an inner and outer membrane with a space called the lumen in between. The inner and outer membranes together are referred to as the nuclear envelope. The outer membrane is continuous with the endoplasmic reticulum. There are pores in the nuclear envelope that allow for the passage of material back and forth
2 categories of chromatin
Heterochromatin and euchromatin
Heterochromatin
Peripheral nucleic acid material. It is compact DNA that usually is not expressed
Euchromatin
Less condensed genetic material, it is more likely to be expressed
What happens in the eukaryotic nucleus?
All nucleic acid originates within the nucleus. DNA is stored, replicated, and transcribed to RNA here. RNA is translated to protein in the endoplasmic reticulum or on free ribosomes
DNA structure
Right handed double helix- there are 2 long polynucleotide strands containing 4 types of nucleotide bases. All nucleic acids are composed of a sugar phosphate backbone and nucleotide bases
DNA base pairing rules
A always pairs with T and G always pairs with C.
Nucleotide
Consists of a 5 carbon sugar (ribose or deoxyribose) with 1 or more phosphates (usually one) and a nitrogen-containing base
Deoxyribose
The 5 carbon sugar found in DNA, which is attached to a single phosphate and nitrogenous base. Missing O2 of deoxyribose allows DNA to form double helix and to coil around histones, allowing for stability. Taking the oxygen away creates less steric hindrance and more flexibility. Makes DNA a more reliable form of information storage. RNA is less stable than DNA. The oxygen is why it is also single stranded
Purines
Adenine (A) and guanine (G)
Pyrimidines
Cytosine (C) or thymine (T)
Deoxyribose vs ribose
Ribose contains an OH group bound to its 2’ carbon, while deoxyribose only has a hydrogen bound to its 2’ carbon. The missing oxygen allows DNA to form double helical structure and coil around histones. Extra oxygen is too close of a Van der Waals force to form the characteristic twisting double helical structure.
Ribose and deoxyribose carbon numbering
Number the carbons going clockwise from the oxygen at the tip of the pentagon. The 5th carbon is part of a CH2 group branching off of carbon 4, which binds to the phosphate group in nucleic acids. The nitrogenous base is bound to carbon 1. DNA synthesizes from 5’ to 3’, referring to the specific carbons
Bonding in a DNA double helix
Double helix formed due to reactions with nitrogenous bases. These Nitrogen bases pair specifically through H-bonds making DNA a double-stranded molecule (The extra oxygen in ribose is enough to make this an unfavorable/unstable reaction). Double-stranded nature makes DNA more stable (more resistant to base mutations) and provides a way in which DNA replication and transcription can be ramped up
Hydrogen bonding between base pairs
Hydrogen bonds are non-covalent bonds and are fairly easy to break. GC has 3 hydrogen bonds, AT has 2 hydrogen bonds, so AT is easier to break apart. Therefore, DNA replication typically begins in areas of DNA that are AT rich
Eukaryotic chromosomes
Long DNA molecules associated with packing proteins. Packing proteins condense down the DNA. We have 23 chromosomes with 2 sets per cell (46 total). There are 22 homologous pairs in males and 23 in females (since females have XX sex chromosomes). Each chromosome consists of many genes- there is a correlation between the number of genes present in a chromosome and organism complexity.
Prokaryotic chromosomes
Chromosomes are circular in prokaryotes, with a few exceptions. Each chromosome has one origin of replication
Prokaryotic origin of replication (oriC)
Each chromosome has one. This is where DNA replication begins. Many origins of replication ensures rapid chromosomal replication
Plasmids
Extrachromosomal prokaryotic DNA that is acquired over evolutionary time. They are usually circular but can be linear. Plasmids are only found in prokaryotic cells, never eukaryotic
Prokaryotic telomeres
Linear plasmid ends that are repeated nucleotide sequences, which allow the ends of the sequence to be replicated. Seal the ends in eukaryotic linear DNA also. Only some prokaryotic DNA is like this. Telomeres seal and protect DNA by preventing chromosome ends being mistaken for broken DNA in need of repair.
Methods condensing prokaryotic DNA (3)
- Loop domain structure
- DNA binding proteins
- Negative supercoiling
Loop domain structure
Many proteins are involved in these structures, forming 30-200 negatively supercoiled loops in a chromosome. It compacts the chromosome from around 15 micrometers to 1 micrometers. DNA binding proteins compact the chromosome further and stabilize this structure
Prokaryotic DNA binding proteins (4)
- HU
- IHF
- H-NS: binds curved/bent DNA, stabilizes
- Fis
HU
A prokaryotic binding protein. It is fairly nonspecific and induces DNA bending
IHF
A prokaryotic binding protein. It binds specific sequences and induces DNA bending
H-NS
A prokaryotic binding protein. It binds curved/bent DNA, stabilizing it
Fis
A prokaryotic DNA binding protein that induces negative supercoiling
Function of DNA binding proteins
They efficiently pack and condense DNA by inducing its bending/folding (in a complex or concave manner). Forming dimers with other proteins is sometimes necessary
Prokaryotic DNA packaging
One DNA loop is composed of DNA associated with many DNA binding proteins, inducing the bends. The binding proteins condense the DNA even further within the loop domains.
Negative supercoiling in prokaryotes
Another DNA condensing strategy that occurs within the individual DNA loop domains. Negative= moving in the left hand direction. Supercoiling within individual DNA loops creates a more compact DNA
How do eukaryotic chromosomes differ from prokaryotic?
Eukaryotic chromosomes are linear, prokaryotic chromosomes are circular
Condensed chromosome vs. interphase chromatin
This chromosome is undergoing mitosis, when it is most condensed. It is usually less condensed under normal conditions
3 specialized chromosomal sequences in eukaryotes
- Origin of replication
- Centromere
- Telomeres
Origin of replication (eukaryotes)
Each eukaryotic chromosome has many origins of replication, in contrast to prokaryotic chromosomes
Centromeres
Kinetochore complex connects this to mitotic spindle for separation of sister chromatids
Telomeres
Repeated nucleotide sequences at the ends of chromosomes, which seals the chromosome ends and allows the ends of linear chromosomes to be replicated. If telomeres are totally depleted, the cell can’t divide any longer. Telomeres protect the chromosome ends and essentially form a seal
Jumping genes
Transposable (mobile) genetic elements found in eukaryotic chromosomes. They are short, non-coding pieces that were acquired over evolutionary time. They may be relevant to packing and structure/storage of DNA, and are found throughout the genome. Most of the eukaryotic genome is non-coding
Genes
Code for proteins (usually, but not always). The average gene size is 27,000 base pairs, with 13,000 base pairs required for the average protein in the eukaryotic cell. Genes can also code for functional RNA molecules. They consist of introns, exons, or regulatory DNA
Introns
Non-coding gene segments in eukaryotes- not found in prokaryotes
Exons
Coding gene segments in eukaryotes- not found in prokaryotes
Regulatory DNA
Sequences that ensure genes are on or off at the proper time, expressed at an appropriate level, and in the proper type of cell. Transcription factors bind to these regions
Nucleosomes
Protein complexes of histones, DNA winds around them. They efficiently pack and condense eukaryotic DNA. Each nucleosome reduces DNA to one third of its original length. There are thousands to millions of nucleosomes condensed with one chromosome.
Compacting of eukaryotic DNA
Eukaryotic and prokaryotic DNA are both compacted, but they are compacted in different ways. Chromosomes are most condensed during mitosis
Histones
The proteins that make up nucleosomes, which DNA winds around in order to condense it. They are unlike DNA binding proteins- do not induce bends in the DNA like is observed in prokaryotes
DNA-winding proteins
Another name for the histone/nucleosome complex in eukaryotes, which differentiates them from DNA-binding proteins found in prokaryotes
Nucleosome structure
The nucleosome protein core is composed of 8 histones, 147 base pair DNA. There are 4 histones present in 2 copies each. Histones H2A, H2B, H3, and H4 (2 each, forming an octamer). There are around 140 base pairs coiling around histones. Each nucleosome core is separated by linker DNA. This linker DNA can be anywhere from a few bp – 80 bp
DNA-histone wrapping
If a nucleosome is opened, you would see 8 histone proteins- H 1-4 present in 2 copies each.
Histone fold
Histones are small proteins that are between 102 and 135 amino acids. Histones share a trimeric structural motif called a histone fold. It is made of 3 alpha helices connected by 2 loops. All 8 of the histones come together in a very specific way to form the nucleosome core.
Formation of a nucleosome (3 steps)
The nucleosome forms in a regulated way
1. The H3-H4 histone tetramer forms and binds to DNA first
2. Two H2A-H2B dimers are assembled. They bind to the tetramer and then the DNA
3. At this point, we have a fully formed nucleosome. Chaperone proteins mediate its assembly
Handshake interaction
2 different histones will come together in a quaternary structure in a handshake interaction
DNA-histone interactions
There is an extensive interface between the DNA and the histones that make up the nucleosome. There are around 142 hydrogen bonds between DNA and the histones that make up an individual nucleosome, and half of these are between the phosphodiester backbones of the DNA and the peptide backbone of the histones. There are also many other non-covalent bonds. Around 1/5 of core amino acids are basic (lysine or arginine) which is perfect for a DNA backbone (single DNA is negatively charged).
Chromatin
Chromatin is a complex of DNA, histone, and non-histone proteins in nucleus. Chromatin stacks in regular arrays (around 30 nm in diameter) to make DNA more compact and save space. X-ray crystallography supports the zig-zag model of chromatin, while cryo-tomography supports solenoidal model. It isn’t well understood which model is correct, but this occurs when neighboring nucleosomes interact with each other
Zig-zag model of chromatin packing
Supported by X-ray crystallography. Does not require a specialized histone
Solenoidal model of chromatin packing
A coiled structure. Supported by cryo-tomography. This model requires a specialized histone called histone H1
N-terminus histone tails
Each protein has an N terminus and a C terminus. There are 8 N termini b/c the histone core of a nucleosome is an octamer. They form tails that “glue” together neighboring nucleosomes and allow them to interact with each other.
Histone H1
A specialized histone that would be present just outside of the nucleosome core. It binds the DNA that exits the histone, acts like molecular glue to glue the neighboring nucleosomes
Heterochromatin
Around 10% of the mammalian cell genome is heterochromatin, which is especially compact. It consists of very few genes since it is not often expressed. The genes packaged here are silenced (this is a positional effect). Heterochromatin is concentrated at centromeres & telomeres – makes sense, as these are not subject to expression like genes
Euchromatin
The opposite of heterochromatin- it is more relaxed and where most genes are found. These genes are more likely to be expressed
Chromatin organization
The DNA double helix wraps around the nucleosome core. The nucleosomes interact with one another to form chromatin. Chromatin can then condense down further
Condensins
Proteins that help to create mitotic chromosomes. During mitosis, the chromosome is the most condensed it will ever be.
Nucleosome dynamics
Nucleosomes are naturally dynamic. Nucleosome DNA unwraps from each end 4 times/sec, essentially rocking back and forth 4 times per second. This allows access for promoters, transcription factors, DNA binding proteins, RNA Polymerase, etc. Can allow for exposure of a promoter on DNA. This is a natural dynamism of nucleosomes
Chromatin remodeling complexes
Protein complexes that bind the nucleosome core and DNA. Facilitates nucleosome sliding, which allows the exposure of promoters and RNA polymerase to bind to promoters, and allows access for DNA binding proteins. It is an active process that requires ATP. Can work with histone chaperones to completely remove the nucleosome core.
Nucleosome sliding
Chromatin remodeling complexes go through ATP hydrolysis. They allow for sliding of the nucleosome, so the DNA unwraps and a promoter or another target sequence is exposed
Types of nucleosome modifications (2)
- Composition of core histones (histone variants)
- Modification of N-terminus tails
These modifications act together to change the histone code.
Composition of core histones
The histones that make up the nucleosome can be swapped out for alternative (less conserved) histones. The swapping out of the histones provides a signal to other things in the cell
Modification of the N terminus tails of core histones
This usually involves the addition of a chemical group to the N-terminal tails on the nucleosome histones. Examples- acetylation, methylation, phosphorylation, and ubiquitination. Different enzymes are involved depending on which chemical group is added.
Histone swapping
In this process, the histones are swapped out for less conserved histones. Histone variants may be inserted into already formed chromatin- this process requires chromatin remodeling complexes. Histone swapping produces an alternate nucleosome that is recognized by host cell machinery
Histone code
The “signal” altered by nucleosome modifications. It is recognized by a code reader complex and associated proteins, which recognizes altered signals. Proteins include the chromatin remodeling complex, which relax the chromatin and lead to its expression
Chromatin remodeling and gene expression
Chromatin remodeling= gene expression. When DNA is relaxed, DNA polymerases and transcription factors have access to the DNA
Chromatin-remodeling complexes
Responsible for histone swapping and work in concert with histone chaperones. This requires ATP
Histone acetyltransferases (HATs)
Transfer acetyl group from acetyl-CoA to the NH3+ (amino group) of lysine, located on the N-terminal tail of the histone
Histone deacetylases (HDACs)
Removes acetyl group with 1 molecule of H2O
Histone acetylation-deacetylation results (3)
- Interactions between neighboring nucleosomes are disrupted, DNA is relaxed
- Acetylation acts as a marker for transcription factors (TFs)
- By adding acetyl groups, the histone code is modified. Chromatin remodeling complexes may be involved to further relax the DNA
Bromodomain
A domain of transcription factors which recognizes and binds to the acetyl group on the lysine. This can stimulate gene expression
Addition of acetyl groups breaking interactions between nucleosomes
The positive histone H4 tail interactions with the negative H2A tail. Acetylation neutralized the + charge, disrupting +/- interactions between H4 and H2a (essentially disrupting the interactions between neighboring nucleosomes and relaxing DNA)
