Lec 3: Basic of genetics: DNA, RNA, Chromosomes, Genes Flashcards
function of chromatin
barcoding in eukaryotic cells
- important for differential gene expression
- basic of phenotypic variability
what are the 4 experiments completed to search for genetic material and determine if nucleic acid, protein/DNA or RNA?
- Griffiths Transformation experiment
- Averys Transformation Experiment
- Hershey Chase Bacteriophage experiment
- Tobacco Mosaic Vitus (TMV) experiment
what did Watson and Crick demonstrate?
double-helix model of DNA
what occurred in 1890 with Weisman?
substance in the cell nuclei controls development
- we can attribute RNA or DNA to what changes the characteristics of that cell
- Know DNA is important in inheritance
what occurs in 1900?
Chromosomes shown to contain hereditary information, later shown to be composed of protein & nucleic acids
Hypothesis of Griffiths Trans. Experiment in 1928
Transfer of genetic information between cells through a process known as transformation
hypothesized that the transforming agent was a “IIIS” protein
- potentially the protein involved
explain Griffiths Trans Experiment
- S. pneumoniae, major cause of pneumonia
- Performed experiment on rats and 2 strains of bacteria that cause pneumonia:
- Type R (rough) = non-encapsulated, avirulent, relatively harmless (NO DISEASE)
- Type S (smooth) =encapsulated, virulent, severe pneumonia
result of Averys Trans Experiment in 1944?
DNA (not RNA, protein fat, or carbohydrate) is the transforming agent
explain Averys Trans experiment
Determined that the DNA from type S bacteria was the genetic material responsible for Griffith’s results (not RNA)
result of Hershey-Chase 1953 Bacteriophage experiment?
DNA (not protein) is the genetic material
what is a bacteriophage?
a virus that attacks bacteria and replicates by invading a living cell and using the cell’s molecular machinery.
virulent T2 bacteriophage is compsed of ____ and ____ ___
DNA and protein shell
- Hijacks bacteria cells and gene expression machinery and uses it for own purpose to build more virulent to have more DNA to multiply and rupture bacteria and affect other cells
describe the 6 stages of lytic cycle of virulent T2 bacteriophage
1) attachment of phage to E.coli and injection of phage chromosome
2) breakdown bacterial chromosome by phage-specific enzyme
3) replication of page chromosome using bacterial materials and phage enzymes
4) expression of phage genes to produce phage structural components
5) assembly of progeny page particles
6) release of progeny phages by lysis of bacterial wall
explain Chase-Hershey Blender experiment 1953
- Set-up two replicates:
(1) Label DNA with phosphorus (32P)
(2) Label Protein with sulfur (35S) - Infected E. coli bacteria with two types of labeled T2
- Blender allowed for the separation of the phage coats from the bacteria
- These bacteria were lysed to release phage progeny. The progeny of the phages that were originally labeled with 32P (DNA) remained labeled, while the progeny of the phages originally labeled with 35S (protein) were unlabeled
results of Grierer and Schreamm/Fraenkel-Conrat and Singer TMV experiment
RNA (not protein) is genetic material of some viruses
Demonstrate RNA is the genetic material of TMV
TMV experimetn
use 2 viral strains (A and B)
- RNA-A is important for the synthesis of protein A
- In TMV-B if take off protein sheath which usually synthesize protein B, and artificially put into sheath of protein A= infect it into the plants and see it still contains B progeny
- Changing the coat doesnt change anything, will have the same coat as the progeny
- Suggests the viral RNA is important for the inheritance and prorogation
results of watson and crick experiment1953
propose double-helix model of dna
The sugar-phosphate backbone is on the outside and the four different bases are on the inside of the DNA molecule
nucleic acids are formed form
nucleotide polymers
name the 3 components of nucleic acids
pentose sugar
nitrogenous bases
P group attached to 5’ carbon
what is a pentose sugar?
5-C sugar
DNA= deoxyribose RNA= ribose
what are nitogenous bases?
purines= Adenine, guanine
pyrimidines= cytosine, thymine (DNA), uracil (RNA)
what is a phosphodiester bond?
Covalent bond between the phosphate group (attached to 5’ carbon) of one nucleotide and the 3’ carbon of the sugar of another nucleotide.
- strong bond
- make DNA stable
what is 5’-3’?
ends of the DNA or RNA chain are not the same. One end of the chain has a 5’ carbon and the other end has a 3’ carbon.
what are the 2 sources of info for the double-helix model of DNA?
1) base composition studies of Erwin Chargaff
2) x-ray diffraction studies
what are base composition studies of Erwin Chargaff?
- indicated double-stranded DNA consists of ~50% purines (A,G) and ~50% pyrimidines (T, C)
- amount of A = amount of T
- amount of G = amount of C (Chargraff’s rules)
- %GC content varies from organism to organism (thermostability)
hot environment= inc GC content in DNA
what are x-ray diffraction studies?
Rosalind Franklin, Maurice Wilkins
conclusion= DNA is helical structure with distinctive regularities , 0.34nm and 3.4nm
what are the 2 main features of double helix model of DNA
1) Two polynucleotide chains wound in a right-handed (clockwise) double-helix.
2) Nucleotide chains are anti-parallel: 5’ –> 3’ amd 3’
major grooves are where ____ dock to parts of DNA to ….
proteins dock
change gene expression
function of ligase?
glue/stick base-pairs
how does DNA replication occur?
- To make a copy of itself, the twisted, compacted double helix of DNA unwinds and separates into two strands
- Each strand becomes a template for making a new strand
- Proper base-pairs are assembled on the template by DNA polymerase
- Nucleotides are connected together by DNA ligase to make a new strand that is identical to the old strand
- new DNA double helix has one strand from the original double helix DNA and one newly synthesized DNA strand
in RNA what are the nucleotide pairs?
A-U
C-G
name 4 characteristics of RNA
- Single stranded and shorter than DNA
- Less stable than DNA
- Ribose is the 5-carbon sugar
- Uracil replaces thymine
4 examples of RNA
mRNA
tRNA
rRNA
snRNA (small nucleus)
- all single stranded, function in transcription (RNA processing) and translation
structural aspects of genome
- Package the DNA in an orderly way
in the cell nucleus.
e.g.,total extended length of DNA in a human cell is ~2 m, but this must be fit into a nucleus with a diameter of ~5-10 μm.
physiological aspects of genome
- DNA is the same in all somatic cells of an organism.
- 25,000 genes è ~140,000 proteins?
- Generate many different cell types (time & space).
- Organize different cells into different tissues/organs and express different proteins.
DNA is transcribed into ???
mRNA
mRNA is _____ into protein
trnaslated
what is the dogma for protein synthesis?
DNA —> [transcription] –> RNA
–> [translation] –> proteins
translation requires 2 things
tRNA and ribosomes
what is the genetic code?
nonoverlapping triplet code
- nucleotides= ATGC
- each 3 base pair codon in gene is transcribed into mRNA and translated to protein
what signals initiation and termination of both transcription and translation?
special sequences
name 3 RNA polymerases
RNA pol I, II, III
what do introns do?
break up genes
what makes eukaryotes different than prokaryotes?
eu=contain chromatin!!!
what is a genophore
chromosome without chromatin
function of chromatin
indexing platform
tells us where genes are
role in folding DNA
DNA of eukaryotic cells is tightly bound to small basic proteins called
histones
DNA is complexed with histones to form….
nucleosomes
each nucleosome consists of how many histone proteins, and wraps DNA how many times?
8 histone proteins
wrap 1.65 times
chromatosome consists of ____ and _____
nucleosome and H1 histone
nucleosomes fold to produce …..
30nm fiber
the 30nm fiber forms loops of how long?
300nm
the 300nm fibers are compressed and produce???
folded to produce 250nm wide fiber
the tight folding of 250nm fiber produces ….
the chromatid of a chromosome
net result of chromatin folding into DNA???
each DNA molecule has been packaged into a mitotic chromosome that is 100,000-fold shorter than its extended length
which histone is an adhesive molecule?
H1
H1 sits outside of nucleosome, why???
important for compaction of nucleosomes
4 levels of packing DNA into chromosomes
1) Winding of DNA around histones to create a nucleosome structure.
2) Nucleosomes connected by strands of linker DNA like beads on a string.
3) Packaging of nucleosomes into 30-nm chromatin fiber.
4) Formation of looped domains.
which histone proteins are inside histone complex?
H1 H2A H2B H3 H4
Enzyme digestion reveals that how many bp of DNA is wrapped around the histone core complex in all cell types
146 bp
describe the charge of DNA vs proteins
DNA= negatively charged
proteins= positively charged
is heterochromatin compact?
yes
heterochromatin transcribed or not?
untranscribed
- no gene expression
- silent genes
what are the 2 types of heterochromatin?
constitutive
facultative
what is constitutive heterochromatin?
- Chromatin that is ‘always’ heterochromatic.
e. g. Telomeres,
(peri) Centromeres.
- prevent chromosome fusion, prevent unravelling
what is facultative heterochromatin
- does not always need to be heterochromatic
- can convert to euchromatin when needed.
e.g., X- chromosome in female mammals (dosage compensation).
is euchromatin compacted or not?
uncompacted
- bead on a string
is euchromatin silent or active?
active
describe ON/OFF and open/closed chromatin
ON= active/open chromatin
OFF= repressed, closed chromatin
what is centromeric DNA?
Center of chromosome
specialized sequences function with the microtubules and spindle apparatus during mitosis/meiosis.
can act as templates for DNA replication
what is telomeric DNA?
- At extreme ends of the chromosome
- maintain stability, and consist of tandem repeats. -
- Play a role in DNA replication and stability of DNA.
what is unique-sequence DNA?
- ‘single copy’
usually code for genes
what is repetitive sequence DNA?-
- interspersed or clustered, vary in size
- can act as tracers, bio-markers for disease
what are 3 types of repetitive-sequence DNA?
SINE= short interspersed repeated seq
LINE= long interspersed repeated seq
microsatellites= short tandem repeats
what are exons?
segment of DNA that codes specific AA (introns do not)
what is transcription
DNA is copied into mRNA with the aid of RNA polymerase.
DNA–> RNA
function of RNA polymerase in transcription
bind to promoters that act as signals in the DNA sequence to make RNA.
what is a way to control gene expression?
control transcription initiation
regulatory proteins
what are regulatory proteins function
- bind to DNA to either block or stimulate transcription, depending on how they interact with RNA polymerase
why do eukaryotic cells regulate gene expression?
to maintain homeostasis in the organism
how do regulatory proteins control gene expression?
bind to specific DNA sequences
- reg proteins gain access to bases of DNA at major groove
- possess DNA-binding motifs
what are DNA-binding motifs?
regions of regulatory proteins which bind to DNA
name 4 types of DNA-binding motifs
helix-turn-helix motif
homeodomain motif
zinc finger motif
leucine zipper motif
what is a motif?
domain of protein responsible for docking protein to major groove
name 4 players in transcription regulation
1) DNA-binding transcription factors (upstream)
2) chromatin regulators
3) coactivators and co-repressors: mediator, etc.
4) basal machinery: RNA PoIII, GTF
what are the 3 large/main steps of transcription?
initiation
elongation
termination
describe the 5 steps in transcription
INITATION
1) polymerase binds to promoter seq in duplex DNA, ‘closed complex’
2) polymerase melt duplex DNA near trans start site
- form trans bubble (‘open complex’)
3) polymerase cataylze phosphodiester linkage at 2 initial RNA nucleotide
ELONGATION
4) polymerase advance 3’–. 5’ down template strand, melt duplex DNA
- add RNA nucleotide to growing RNA
TERMINATION
5) at trans stop site, polymerase release completed RNA and dissociates from DNA
list the 6 broken down trans initiation steps
- promoter (start site) recognition
- promoter binding
- promoter melting
- transcript initiation
- promoter escape/clearance
- transcript elongation
controlling expression of eukaryotic genes requires what???
transcription factors
2 types of Tc
general Tc
specific Tc
what is general Tc required for?
- are required for transcription initiation
- required for proper binding of RNA polymerase to the DNA
function of specific Tc?
inc transcription in certain cells or in response to signals
what is the TA-TA region?
where RNA polymerase binds
describe eukaryotic gene transcription
- general Tc bind to promoter region of gene
- RNA polymerase II then binds to the promoter to begin transcription
at the start site (+1) - Enhancers are DNA sequences to which specific transcription
factors (activators) bind to increase the rate of transcription - only when transcription factors bind to the promoter region that RNA polymerase is placed in an orientation that allows the initiation (start) of transcription
____ and ____ are also required for function of Tc?
coactivators
mediators
function of coactivators and mediators?
bind to Tc and other parts of the transcription apparatus
what are general factors?
B,F, E
- Tc
- position RNA polymerase at start of a protein-coding seq, then release polymerase for initiation
what happens if have mutation?
- change abundancy of gene transcription (each Tc have particular domains bind to DNA)
- DNA can block and include Tc
- create binding sites on promoter that didnt previously exist, Tc can properly activate gene now
describe 2 general ways to alter with phenotypes
1) altered structure
- variation in expressed seq
- altered mRNA, protein
- normal levels
= altered phenotype (diff protein function)
2) altered expression
- variation in regulatory sequence
- normal mRNA, normal protein
- altered levels
= altered phenotype
if change the exon sequence by 1 nucleotide leads to … (3)
- Prevention of transcription
- Prevention or incorrect processing of mRNA - i.e., can’t make a protein
- Protein with reduced/absent function or different function
what is a mutation?
changes in the DNA sequence passed on to future generations
what is a point mutation?
a single base substitution
e.g., Single Nucleotide Polymorphism (SNPs) occur commonly within a population (i.e., 1%)
name 3 types of point mutations
silent
nonsense
missense (inappropriate synthesis of AA)
what is frame-shift mutation?
modification of the reading frame after a deletion or insertion,
resulting in all codons down stream being different (i.e., the codon sequence is shifted)
what is substitution?
a different nucleotide is substituted
e.g. Sickle Cell Anemia
what is insertion?
the addition of a new nucleotide
e.g. Huntington’s Disease)
what is deletion??
the loss of a nucleotide
e.g. Tay-Sachs Disease
what is PKU an example of???
insertion
what is PKU??
- Gene mutation, insertion of premature stop codon
- autosomal recessive
- Genes are not expressed to make the liver enzyme phenylalanine hydroxylase (PAH)
- Cannot convert the essential amino acid phenylalanine (Phe) to tyrosine, which is the
precursor of the neurotransmitters dopamine and norepinephrine - If left untreated, phenylalanine builds up in the body to toxic levels, leading
to progressive developmental delay, irreversible brain damage, severe mental retardation, seizures, autistic-like behaviors, a peculiar (musty) odor, hyperactivity and eczema, and excretion of large amounts of phenylalanine in urine
what is chromatin remodeling?
- necessary for transcription
- nucleosomes block RNA polyerase II from gainign access to promoters
- need to decondense DNA to open
- addition of acetyl groups to histone tail remodel the solenoid so DNA is accessible for transcription
- during initiation, nucleosome complexes unravel–> by remodeling protein
what is function of Swi/Snf complex?
removes nucleosomes and deposits histone varients (e.g., H2AZ) for specialized functions (e.g., heterochromatin).
how to dissociate protein/DNA
use high salt conditions
- release 146 bp from protein complex (octamer, 4 duplicate units)
what is the core octamer?
[2x] H2A, H2B, H3, H4
major proteins of chromatin are….
histones
what are histones?
- small proteins contain high proportion of pos charged amino acids (arginine, lysine) that facilitate binding of neg charged (acidic) DNA molecule
name the 5 major types of histones
H1 H2A H2B H3 H4
how does histone fold?
- fold into handshake structure
- dimers form with protein, heteromers (4 proteins) then 2x4 proteins is octamer, fit inside loop of 146bp of DNA
what are the 4 main functions of chromatin????
1) pack DNA into small volume
2) strengthen DNA to allow mitosis
3) prevent DNA damage (protect at heterochromatin end)
4) control gene expression and DNA replication
