Part 4 Flashcards
The Human Genome Project was completed in what year?
2003
As organism complexity increases……
- genome size (the # bp) increases
- # of genes increases
- gene density decreases dramatically (average # of genes per Mb of genomic DNA)
E.coli genome is composed almost entirely of genes. T/F
- genome: 4.6 Mb
- single chromosome
- composed of encoding proteins or non-coding RNAs and most non-coding sequences are involved in gene regulation
The amount of DNA used to encode the RNA polymerase large subunit gene is highly variable, despite similar size of the resulting polypeptide product.. T/F
True
The amount of DNA used to encode the RNA polymerase large subunit gene is highly variable, despite similar size of the resulting polypeptide product.. T/F
True
What are the two reasons why there is a decrease in gene density?
- Increases in gene size, due to the presence of introns that eventually get removed from mRNA following transcription via RNA splicing
- Introns can dramatically increase gene length
* only 5% of an average human protein-coding gene is coding for an amino acid sequence (95% introns) - intergenic sequences
- more than 60% of the genome- much has no known function
- 2 types: unique and repeated
Unique intergenic sequences
25% of intergenic DNA
- regulatory sequences: regions of DNA required to direct and regulate gene expression. There are more regulatory sequences required to coordinate proper gene expression in complex multicellular organisms with more genes
- Nonfunctional genetic artifacts: nonfunctional mutant genes or gene fragments
- microRNAs (miRNAs): regulates gene expression
More potential functions of unique intergenic sequences are not yet discovered
Repeated internecine sequences
75% of intergenic DNA
- microsatellite DNA
- very short sequences less that 13 bp that are tandemly repeated
- almost 3% of the human genome
- most common are dinucleotide repeats
- also called VNTRs: (variable number tandem repeats) because different individuals in the population have variable number of repeats of a short nt sequence at different loci in the genome. This is used in genetics, forensics, paternity testing, etc. - genome-wide repeats
- between 100-1000 bps
- present in the genome as either a single copy or closely spaced clusters
- 43% of the human genome
- all are transposable elements: this is how they have become such a huge part of the genome
- stable maintenance over thousands of generations
What is a transposable element?
- sequences that can move from one place in the genome to another
- the element is either excised and “jumps” to a new position, or a copy of the element moves to a new position (multiplication)
What are chromosomes?
- cellular DNA is associated with proteins and arranged in chromosomes
- prokaryotes, eukaryotes, and viruses
What are some functions of chromosomal arrangement of DNA?
- compacts DNA to fit inside the cell
- protects DNA from damage
- Transmitted efficiently to daughter cells during cell division
- regulates the accessibility of DNA- serves as a level of regulation for gene expression
What are some functions of chromosomal arrangement of DNA?
- compacts DNA to fit inside the cell
- protects DNA from damage
- Transmitted efficiently to daughter cells during cell division
- regulates the accessibility of DNA- serves as a level of regulation for gene expression
What is the chromosomal makeup of prokaryotes and eukaryotes?
prokaryotes: single copy, circular
eukaryotes: 1 or 2 copies, linear
Bacteria
- Consists of: nucleoid, chromosomes, plasmid, capsule, and flagellum
- typically one complete copy of the chromosome packaged into “nucleoid” region
- extrachromosomal DNA plasmids: present in multiple copies; often carry genes that are not essential for survival but confer desirable traits
Viruses
- simplest living organism dependent on host cell
- unlike bacteria, archaea and eukaryotes, viral genomes come in many different forms:
1. DNA or RNA
2. double or single stranded
3. linear or circular
4. one or multiple copies - can be 2,000 to 1,200,000 bp in size
Retroviruses
- single-stranded RNA viruses
- contain REVERSE TRANSCRIPTASE that can produce DNA from its RNA genome
- insert a DNA copy of its genome into the host cell in order to replicate
- has an envelope
ex: HIV and HTLV
Retroviruses
- single-stranded RNA viruses
- contain REVERSE TRANSCRIPTASE that can produce DNA from its RNA genome
- insert a DNA copy of its genome into the host cell in order to replicate
- has an envelope
ex: HIV and HTLV
Eukaryotic Cells
- chromosomes are always contained within a membrane bound nucleus
- have multiple linear chromosomes in the nucleus
Length of DNA all laid out
1 meter
Diameter of an average human cell nucleus
0.00001 to 0.000015m
What is a nucleosome?
The building blocks of eukaryotic chromatin
DNA wrapped around a protein histone core
-The first level of DNA packaging: compacts the DNA 6-fold!
chromatin
Beads (nucleosomes) on a string (linker DNA)
Histones
- most abundant protein associated with eukaryotic DNA
- relatively small proteins
- basic
- positively charged
- more than 20% of the aa in each histone are lysine and arginine because they associate tightly with negatively charged DNA
What are the 5 abundant histones?
H1, H2A, H2B, H3, H4
What are the 3 core histones?
H2A, H2B, H3, H4
H1
Linker histone
*there is only 1 per nucleosome
What are the 5 abundant histones?
H1, H2A, H2B, H3, H4
The 3 in the core are: H2A, H2B, H3, H4
H1: Linker histone
*there is only 1 per nucleosome
What are the 5 abundant histones?
H1, H2A, H2B, H3, H4
The 3 in the core are: H2A, H2B, H3, H4
H1: Linker histone
*there is only 1 per nucleosome
Core histones in the Nucleosome make an _____.
octamer- 8 polypeptides
There are 2 copies of each, per nucleosome H2A H2B H3 H4
Core histones share a common structural motif.
histone-fold domain mediates assembly of histone protein core of nucleosome (in absence of DNA)
3 alpha helices are separated by unstructured loops
Core histones share a common structural motif.
histone-fold domain mediates assembly of histone protein core of nucleosome (in absence of DNA)
3 alpha helices are separated by unstructured loops
Nucleosome Assembly
- H3_2-H4_2 tetramer binds to dsDNA
- 2 H2A-H2B dimers are recruited
Final assembly of nucleosome-leaves accessible histone “tails” (amino-terminal extensions). N-terminal tails of core histones are accessible to enzymes.
What happens to N-terminal tails of core histones?
- treatment of nucleosomes with a protease trypsin
- Trypsin specifically cleaves proteins after positively charged amino acids such as Lys and Arg
- Trypsin treatment rapidly removes the N-terminal tails but leaves the histone core intact
- modifications of the Histone amino-terminal tails alters chromatin function. N-terminal tails are the site of extensive post-translational modifications that alter the function of individual nucleosomes
- acetylation
- methylation
DNA-histone interactions demonstrate _______.
non-specific binding
histone proteins mainly contact DNA through IONIC BONDS (backbone) and H-BONDS (backbone or minor groove) which are sequence non-specific
Linker Histone H1
- interacts with DNA at two regions
1. linker DNA at one end of the nucleosome
2. middle of the wound nucleosomal DNA - this interaction further tightens the association of the DNA with the nucleosome
- there is only one H1 per nucleosome
Linker Histone H1
- interacts with DNA at two regions
1. linker DNA at one end of the nucleosome
2. middle of the wound nucleosomal DNA - this interaction further tightens the association of the DNA with the nucleosome
- there is only one H1 per nucleosome
What is the second level of DNA compaction?
- histone H1 interaction with DNA
- gives it a defined structure (distinct zigzag appearance)
- binding of H1 stabilizes higher order chromatin structures (30nm fiber) which makes DNA less accessible. N-terminal tails are required for the formation of this
1. interact with adjacent nucleosome
2. histone tail post-translational modifications may influence these interactions
Different structures of chromatin:
naked DNA
10 nm fiber
30 nm fiber
naked DNA
10 nm fiber
*regions of DNA that are being actively transcribed or replicated are in this form
30 nm fiber
*extensive, additional folding of the 30 nm fiber is required to compact the DNA further to fit into the cell
Nucleosomes store ______ supercoils.
negative supercoils
- DNA wraps around the histone octamer in a L-handed manner which introduces negative supercoils into the DNA
- if the nucleosomes were removed from the DNA, the DNA would be negatively supercoiled
- provides increased access to DNA for DNA replication and transcription and promotes unwinding
Do prokaryotes have nucleosomes? What do they have instead?
No prokaryotes do not have nucleosomes. Instead they have DNA Gyrase.
DNA Gyrase
A specialized topoisomerase that actively introduces negative supercoils into realized DNA; requires ATP hydrolysis
***in prokaryotes
DNA Gyrase
A specialized topoisomerase that actively introduces negative supercoils into realized DNA; requires ATP hydrolysis
***in prokaryotes
What are the 2 forms of writhe for supercoiled DNA?
- interwound
2. toroidal/ spiral (like a slinky); occurs when DNA wraps around the histone octomer
Topoisomerase
unbound DNA is relaxed by topoisomerase and it decreases the linking number
What are the two major categories that chromosomes are divided into?
- Heterochromatin
2. Euchromatin
Heterochromatin
- first characterized by a dense staining seen in a light microscope
- more condensed chromatin
- areas with limited gene expression
- high-order, large nucleosome assemblies
Euchromatin
- poor staining with dyes in the light microscope
- relatively open structure
- higher levels of gene expression
- less organized nucleosomal assemblies (not naked DNA)
