DNA Synthesis: S Genes 09/23/2013
F Chromosomes are made of a combination of a DNA strand and two copies of each (from each
parent). They look that way because each DNA molecule is wrapped around proteins called
histones and knots up to form a condensed chromosomes.
The DNA is formed as a double helix with a sugar phosphate backbone and used complementary
base pairs joined by hydrogen bonding (T, A, G, C)
T and A are always paired together and the same goes for G and C.
DNA replication strands are semiconservative meaning that they go in the opposite direction
forming two strands (one is new and the other is an original based on the base pairing rules).
These strands are antiparallel and the sugar strands consist of carbonated phosphate that attaches
to the backbone of the DNA.
The DNA molecules are surprisingly flexible and when ribose sugars bends it’s bonds you will
either get a chair conformation or a boat conformation.
The critical feature about forming DNA is forming the deoxyribonucleotides (no oxygen) and in
RNA oxygen is present.
DNA polymerase builds new strands of DNA, enforces base pairing rules, and creates new
It will only work when you have a strand of DNA and then one 3 prime hydroxyl with some of
the template missing. The primes are the number of carbons in the sugar. This allows DNA
synthesis to start and the primers are normally RNA not DNA.
DNA synthesis only happens when the strands separate and this happens in multiple places on
the double helix (origins of replication, used in the S phase of the cell cycle).
Helicase separates the strands of the double helix
Single strand binding protein – stabilizes DNA and keeps them from binding back together
Topoisomerase – relieves unwinding tension in the double helix by breaking one of the bonds of
the two strands and flips it to relieve the tension and then connects it as the tension leaves.
Primase – builds the RNA primer (made of 810 bases) to provide the initial 3 prime hydroxyl
Leading strand is responsible for starting DNA synthesis and this is called continuous DNA
The lagging strands is responsible for going in front of the leading strand and then connect back
to the leading strand.
DNA polymerase III 5' to 3’ synthesis (primer) builds off the hydroxyl
DNA polymerase I – chew out the RNA of the primer and fill it in with DNA.
DNA ligase – creates the final bond closing off DNA synthesis. Cancer 09/23/2013
Hallmarks of Cancer
Self sufficiency for growth signals can turn on and keep the cell on activating the division
Failure to respond to growth control signals
Sustained angiogenesis they can grow their own blood supply (by branching the blood vessels
nearby into them in order to get oxygen and nutrients stopping oxygen/nutrient diffusion).
Evasion of programmed cell death
Rebuilds the end of their enzymes in order to keep growing.
Limitless replicative potential
Some cancer cells when dividing can leave behind chromosomes and others can make 4 poles
when getting to the metaphase and anaphase checkpoint instead of the normal 2.
Over replication of the chromosomes have a mess of the genes and these cause major problems
in the cell.
Tissue invasion and metastasis
Metastasis – cancer cells that have spread from the initial site either through the blood stream or
through the rest of the body
Genomic instability/high mutation rate
Don’t copy chromosomes incorrectly and their genomes are wrong as well.
Resistant to treatment – through mutation to the chemotherapy agent
Cachexia – cancers of the esophagus and the lungs cause a wasting disorder where you are
unable to eat and gain the nutrients you need; you literally waste away and end up looking
Lung cancer patients have clubbing of the fingers and cachexia.
Remember that cancer is not just limited to humans but goes to other mammals.
Sun cancer – different proteins that are responsible for repairing damage from harmful UV rays
(9 central genes) and when exposed to sunlight the genes are unable to repair the damage. This
type of cancer is inherited.
Double strand break of chromosomes
It is identified by proteins that stick to the broken ends Cancer 09/23/2013
Nonhomologous ends – ends that are near each other just rejoin to one another even though
they are wrong.
Homologous – recombine the correct chromosome to the other one and keeps the chromosome
more or less the same
P53 is responsible for this type of repair
Benzo (a) pyrene – potent carcinogen that is responsible for lung cancer. Mutating the DNA
genes and targets specific places on the chromosome does this. (Nonrandom mutation).
Base 157, 248 and 273 are the favorite places of this carcinogen.
Most cancer cells will target p53 first because it is responsible for programming cell death or
DNA repair. PCR 09/23/2013
Plasmids – used in the cloning of DNA fragments and they contain multiple cloning sites (MCS)
MCS – they can be cut open with restriction enzymes and DNA fragments are then inserted into
Ex. pUC19 cloning site that contains BamH1 site and EcoR1 site. (3, 686 bp long)
In cloning there are individual molecules that you can make many copies of them and when put
back into the MCS they can be put into different orientations depending on the way it was put in.
PCR – amplifies DNA fragments Transcription factors and Enhancers 09/23/2013
Endo16 – gene that contributes to the formation about a sea urchins gut and one of the first gene
expressions we have seen. Expressed early in time and space.
To do transcription, the DNA strands have to be separated in order to provide a template and the
RNA polymerase then creates a new molecule using the base pairing rules and references the
To build the RNA molecule you must have phosphodiester bonds using either the 5’ or 3’ base
RNA polymerase – separates the DNA strands and builds the phosphodiester bonds using the
base pairing rules. It will only make one strand!
Arthur Kornberg – won the Nobel Prize for figuring out DNA polymerase. (1959)
Roger Kornberg – Arthur Kornberg’s son won the Nobel Prize for RNA polymerase (200?)
The promoter or regulatory sequence is responsible for making binding sites for various
Combinatorial regulation or transcription
Multiple transcription factors are required for gene expression
Transcription factors activate or block gene expression in specific combinations
DNA sequences of promoter regions of genes, and the presence of absence of specific
transcription factors determined by gene expression.
Promoter fusion gene – promoter changes the location
Tetra cyclin – controls transcriptional activation
HIV integrates into the cells, something you can also use to change DNA transcription
Position effects – transcribed regions are controlled by where they are located. Regulation of Gene Expression 09/23/2013
Combinations of DNA can be regulated together since they share the same binding sites and is
mostly based on the DNA structure (sugar ribose backbone).
RNA falls off of the DNA and stops transcribing by forming a structure called a stem loop.
Stem loop – the space between the two structures so that the base pairing sequences are perfectly
arranged. When this forms its interacts with RNA polymerase and causes it to fall off of the
DNA. (TRANSCRIPTION STOP SIGNAL)
UTR – UN translated region, located in the RNA.
Upstream of the UTR – is not located in the UTR and downstream.
Exons are glued together remain in order to form the rest of the RNA strands
Introns are the sequences in the primary strand and are removed from RNA – form the 3’ end and
those ends create the binding site in order to remove it from the RNA. The 5’ end of it is called
the donor site and the 3’ end is called the acceptors.
SnRNPs are the editors of the RNA by binding to the donor and accepting site forming a laurite
and splice out the intron site. Needs a special base, the A nucleotide, is responsible for this and
then joins together the exon end.
Needs a special double phosphodiester bond that joins the two exons together. RNA Processing Part II 09/23/2013
RNA Processing Basic Elements
Exons – the coding regions of RNA normally located at the ends of the DNA strand/fragment.
(5’ to 3’)
During alternate RNA processing differing exons can be connected together to form a laureate
(loop). In order to block a particular exon a blocking site on a donor site on the introns is formed.
Then the snRNP moves downstream to the next donor site.
Introns – the noncoding regions of RNA are located in between the exons and are eventually
removed from the fragment in order for the exon ends to be bound together and create a double
helix. (5’ to 3’)
Typically the intron being with the GT sequence and end with the AG sequence (the GTAG rule)
Splicing – mRNA processing removes the introns from the DNA fragment and splices or
attaches the exon ends together.
SnRNPs – the editors of the RNA. They work by binding to the donor and accepting sites,
forming a laurite (double bend or loops) and splice out the intron site.
A special base called the A nucleotide, is responsible for this and joining together the exon ends.
Needs a special double phosphodiester bond that joins the two exons together
In fruit flies the transformer gene – sex determination gene in flies, causes a female to look and
behave as males except for the fact that they are sterile (XX chromosomes instead of XY
The RNA in XX flies were smaller than in XY flies and they are stopped in the middle of RNA
processing in order to delete a specific gene since a gene called SXL (sexlethal) binds to a
protein and intron during alternative RNA processing
Promoter elements – elaborate mechanisms for regulating the initiation of transcription, which
is mainly, used on the different RNA polymerases (I, II, III).
RNA polymerase I promoter – makes RNA molecules that need to be fairly constant at all
RNA polymerase II promoter – recognizes a variety of promoter sequences and plays a role in
controlling gene expression and the rates of specific gene expression.
RNA polymerase III promoter – makes RNA molecules that need to be fairly constant at all
TATA box – at transcription start site that is 5’ – 3’ in nature and goes like: 5’TATAWAW3’ –
happens in transcription and allows transcription to happen.
Regulatory Protein Binding Sites RNA Processing Part II 09/23/2013
Transcription factors – positive regulators of DNA transcription they are sequencespecific
DNA binding proteins. Through the use of enhancers or silencers, they regulate gene expression
Constitutive transcription factors – promote many different genes and do not seem to respond
to external signals
Regulatory transcription factors – promotes a limited number of genes and responds to
RNA polymerase II transcripts known as hnRNAs, (highmolecularweight nuclear RNAs or
heterogeneous RNAs) are "processed" into mRNA by the steps:
Capping chemical alterations (including methylation) at the 5' end of all hnRNAs
Splicing precise removal of introns from the interior of hnRNAs
Polyadenylation process of replacing the 3' end of an hnRNA with a stretch of approximately
250 A's (the "polyA tail")
Protein is made of chains of amino acids that fold up and can act as many things such as
collagen, keratin, kinases, receptors etc.
Amino acids (20 different ones) all have the same common cores, two abbreviations that are
either 3 letters or single letter code, which makes things easier for them.
Four types of charges: basic – positively charge, or acidic – negatively charged, polar – positive
or negative charge, and nonpolar –no charge given off – neutral.
Bonded together with a peptide bond.
Also they are formed from 5’ to 3’.
Helixalpha helicases – single strand of helix amino acids that form beta sheets.
Translation making a sequence of amino acids in proteins by using the transfer RNA (tRNA)
through the use of messenger RNA (mRNA). By using anticodons it allowed to make sure that
the translation is correct.
Aminoacyl tRNA synthetase – is responsible for the correct matching of amino acids to RNA.
Makes sure that each amino acid has the correct RNA match.
Ribosomes – stick anticodons to the RNA and make sure that the tRNA is connected together in
order make sure that translation is regulated correct
Phases of Translation
Initiation phase – free RNAs floating around and the ribosome binding sites are located on the
RNA. The smaller subunit of ribosomes then connects to the binding site located on RNA. The
start codon is always AUG and specifies amino acid. Then a large subunit of ribosomes binds to
the RNA and translation begins. RNA Processing Part II 09/23/2013
Elongation phase – incoming Aminoacyl RNA – new tRNA moves into A site where its
anticodon base pairs with the mRNA codon. The peptide bond is then formed through the use of
the P site. The ribosomes are then trans locating down mRNA.
Termination phase – encounters a stop codon (UAGUGAUGG). This causes the receptor to
fall off and the signal to stop.
Opening reading frame – the distance between the start (AUG) codon and the stop codon, read
Frame shift mutation – the mutations that cause deletions and additions in the reading frame of
base pairs causes a shift frame. (No longer a 3x3 read)
Missense mutation change in the nucleotide sequence where the bases are changed – they are
still the same size however – relating to RNA sequencing – and that leads to a change in the
amino acid (size wise – bigger or same size)
Nonsense mutation – change a codon that encodes a particular amino acid into a stop codon
causing translation to be cut short.
Silent mutation – a single change in a nucleotide (DNA and RNA change) but nothing happens
to the amino acid. The amino acid is still the same but the DNA and RNA are different.
You can regulate the level of translation by the stop codon or the binding sites. Regulation of Gene Expression 09/23/2013
Regulation Gene Expression
DNA is wound around histones creating chromatin
Condensed chromatin – causes chromatids to be deeply condensed with no binding sites out for
Histone acetyl transferase – HAT adds acetyl groups to histones, opening the chromatin
structure and allowing genes to be turned on.
Histone deacetylase – HDAC removes acetyl groups, condensing the chromatin structures
turning off genes.
DNA methyl transferases – turn on HAT and keep HDAC from turning on.
Acetyl groups removed charged parts of DNA and without them chromatins would attract and
RNA Interference (RNAi) – short double stranded RNA that becomes single stranded and can
eventually base pair with messenger RNA when bound to messenger RNA, it is degraded and
then translation stops. Happens after the RNA is fully processed.
MiRNA – micro RNA found naturally in the body and is responsible for taking care of RNA
problems. (Turns off genes)
Used for purposely turning off certain genes in biotechnology.
SiRNA – short interfering RNA binds to mRNA and destroys it.
There are differing combinations that are used for shutting off gene expression either in the
treatment of disease or for science purposes.
CKDI binding protein – when present CDKI kinases aren’t processed correctly and then are
eventually turned off.
Nuclear pore complex – form holes that act as a way for things to get out of the nucleus into
either the cytoplasm or out of the cell completely.
Protein that has 18 bp long and their destination is going to the nucleus through the nuclear pore.
Nuclear localization signal – it is required to move proteins from the cytoplasm into the nucleus
and binds to Importin in order to move into the nucleus.
Part of nuclear genes
Some transcription factors ability to move into the nucleus is dependent on their ability to bind to
Endocytosis – vesicles containing signal proteins that fuse with the membrane and release the
signal outside of the cell.
Important in nerve cell signaling. Regulation of Gene Expression 09/23/2013
Process: signal sequence (a few amino acids) directs translation to the membrane ▯starts with the
ER and recognizes by SRP and then with the use of SRP it recognizes the docking protein on
RER. ▯The signal sequence allows it to be docked to the membrane ▯go through the membrane
and then stick inside the membrane.
Other ways to have protein stick to the membrane but not all the way through (receptors): having
the signal sequence be a little way through and be stuck or have a stop transfer signal during the
middle of the membrane.
Signal recognition protein – brings the proteins with the signal sequence to the membrane so that it
can be pushed out of the cell. 10/18 Quiz Discussion: Basic Topics 09/23/2013
Nuclear localization signal (NLS) is six amino acids that are part of the protein and is
controlled by translation – in the coding region (cannot be in the UTR region)
The signal sequence consists of translated proteins and it is located at the very front end of the
protein, and cannot be in the UTR region
Both of these are affected by frame shift mutations because it changes the types of amino acids
made – assume that the protein is shorter 90% of the time.
Frame shift mutations in introns – DOESN’T EXIST
Both are affected by coding region mutations
Transcription – RNA polymerase and hairpin loops ▯ RNA processing – when introns are
removed and snRNPs binding sites are used ▯ Translation – frame shifts only affect translation
and are in the coding region the intron is removed (happens in the cytoplasm)
SnRNPs binding sites (donor sites – 5’ end) can be messed up by adding and removing intron
sequences, which means that either nothing happens or the snRNP is hit and the intron cannot be
By blocking acceptor sites, the donor site goes to the next one and acts like the one it skipped is
an intron and then it is removed. (Smaller mRNA)
If the donor site is blocked, nothing is scanned for removal and then the intron never gets
removed (bigger mRNA)
Untranslated regions – are located before and after the coding regions (5’ – 3’)
Mutations in them affect only the size of the RNA and the ribosome binding sites
Ribosome binding site – is located in front of the stop codon in the 5’ UTR that allows protein
to be made.
Hairpin loop – stops transcription – RNA polymerase falls off – happens just before end of the
exon at the 3’ UTR.
Not affected by any of the mutations in translation
Okazaki fragments – are joined together with DNA ligase and are at the end of the lagging
strands in RNA processing
TATA box – 5’ prime of the AUG start codon meaning it’s in the very beginning of the protein
Receptors – starts the signal sequence to become with the membrane and then encounters a stop
signal sequence allowing it to stop in the middle of the membrane and then the rest of the protein
is made outside of the membrane.
All synthesis of nucleic acid is added from the 3’ end.
Some combination of genes that allow genes to divide (look at the diagram)
B and C are the binding sites on the sheet
Signaling pathways cause various cells to divide. 10/18 Quiz Discussion: Basic Topics 09/23/2013 Protein Structure 09/23/2013
Proteins do the bulk of everything in the cell
Proteins form different structure because of the differing sequences of amino acids.
Ex. TATA binding protein
Proteins can change function and shape when phosphorylated
Ex. IRK – active or inactive depending on phosphate attachment to the activation site.4x
Start out with the primary structure of amino acids ▯helix or bet sheets
By folding up on each other they form tertiary structures and the ones that quaternary
structures are multiple proteins that are put together.
When they are misfolded or folded in different ways, they can either work or not work.
Some amino acids are negatively charged since the molecules are unequally sharing them and the
ones that do not have a lot of electrons are positively charged.
Ex. Oxygen (partially negatively charged)
Amino acids are bonding together using peptide bonds ▯amino group at th