Exam 3 Micro Lecture: Ch 8 DNA and Genetics
The basic unit of nucleic acids
Nucleotides are made up of 3 parts:
1. phosphate group
2. 5 carbon sugar (deoxyribose sugar)
3. nitrogenous base
The Central Dogma (3 parts)
DNA makes RNA (transcription) and RNA makes protein (translation)
1. DNA is copied to DNA (Replication - DNA synthesis)
2. DNA information is copied into mRNA (Transcription - RNA synthesis)
3. Proteins are synthesized using the information in mRNA as a template (Translation - protein synthesis)
a macromolecule composed of repeating units called nucleotides
exists in the cell as long strands of nucleotides twisted together in pairs to form a double helix.
The 4 nitrogenous bases that make up DNA neucleotides
How do the pairs line up to form the double stranded DNA helix?
Guanine (purine) always pairs with cytosine (pyramidine) and adenine (purine) always pairs with thymine (pyramidine).
The two strands of DNA are complimentary, not identical
How is the DNA helix held together?
It is covalently held with hydrogen bonds
The double helix unwinds and then complementary nucleotides are matched up to the exposed bases on both strands of the original DNA.
Each DNA strand is a template for the synthesis of a new strand of DNA. After both strands of DNA are replicated, each original DNA strand rewinds with a newly synthesized strand.
the original strand of DNA used to match up nucleotides
the enzyme that pulls the DNA double helix apart into two strands
Why is DNA replication called semiconservative?
Because both new double-stranded DNA molecules contain one original strand that was preexisting and one new strand. The process of DNA replication is referred to as semiconservative replication.
What happens after the DNA has replicated?
the cell divides, forming two daughter cells, each containing identical genetic information
provide the instructions for making specific proteins
The bridge between DNA and protein synthesis
How is RNA similar to DNA?
It is similar in all ways except its sugar is ribose and in place of thymine, there is uracil
the information stored in a DNA molecule is copied into RNA molecules
During transcription, a strand of RNA is made using a specific gene - a portion of the cell's DNA - as a template.
Just like in DNA replication, the DNA must first unwind. Then, complementary RNA nucleotides are matched up to the exposed bases on one strand of DNA. G pairs with C and A pairs with U in RNA transcription.
When transcription of a gene is finished, RNA is released from the DNA
Depending on the gene, transcription makes one of three possible kinds of RNA:
1. messenger RNA (or mRNA)
2. ribosomal RNA (or rRNA)
3. transfer RNA (or tRNA)
Messenger RNA carries the coded information blueprint (the message) from the DNA to the ribosome for the making of proteins.
The source of information.
Ribosomal RNA forms ribosomes, the site of protein synthesis
Makes sure that we start in the right spot and catalyzes the reactions
Transfer RNA bring amino acids (specific amino acids dictated by the sequence of codons) to the ribosome where they are incorporated into proteins
functions as an interpreter between the language of nucleic acids and the language of protein
RNA that has been folded up into a U shape
lines everyone up
decoding the language of nucleic acids and converting it into the language of proteins to make protein
ribosomes use the mRNA produced by transcription to direct the synthesis of a protein following the genetic code
we are changing molecular languages. You are going from nucleic acid language to amino acid language.
The sequence of amino acids gives us the primary structure of the protein. Then we fold that up into a shape so that it can function as whatever that sequence needs to do (a structural protein, a motor protein, an enzyme, etc)
The rules that govern translation
What does the order of the bases in mRNA specify?
The order of the bases in mRNA specifies the order of amino acids for a particular protein.
a sequence of three DNA or RNA nucleotides that corresponds with a specific amino acid or stop signal during protein synthesis
How are the bases on the mRNA molecule are "read" or translated?
As sequential codons. Three DNA nucleotides will be transcribed into one RNA codon. And one RNA codon will be translated into one amino acid.
The key players in the process of translation are what?
ribosomes and transfer RNA (tRNA)
a three-nucleotide sequence that will match up to a complementary codon on the mRNA to form amino acids
How is tRNA structured?
tRNA carries a specific amino acid at one end, and at the other end it has an anticodon
What is the function of the ribosome?
The function of the ribosome is to direct the orderly binding of tRNAs to codons and to assemble the amino acids into a chain, ultimately producing a protein
It is the workbench where protein is constructed from amino acids.
The ribosome is the place where all the necessary components to make a particular protein come together
Translation begins when?
when the mRNA attaches to a ribosome
What are the 6 steps in translation?
1. The small subunit of the ribosome binds to the mRNA, (to that message) followed by the large subunit and gets it lined up so that it can find the start codon
2. The first worker tRNA carrying the first amino acid attaches to the first codon.
3. Next, a second worker tRNA moves into position on the mRNA and sits next to the first tRNA. The second tRNA can only sit on the ribosome if it carries an anticodon that is complementary to that codon.
4. Now that everything matches correctly, the ribosome catalyzes the transfer of the amino acid on the first tRNA to the amino acid on the second tRNA. A peptide bond forms between the two amino acids and --voila!-- we have started building a protein.
5. But, since proteins are made of hundreds of amino acids connected together, this process has to repeat over and over. The ribosome keeps moving along the mRNA from one codon to the next. As the proper amino acids are brought into line one by one by tRNAs, peptide bonds are formed between them, and a polypeptide chain forms.
6. When a ribosome reaches a stop codon on mRNA, the newly synthesized protein chain is released from the tRNA. Translation is now complete.
a group of genes that are regulated in a coordinated fashion
operons are either ______ or ______
the genes are in the "off" mode until an inducer is present which acts to induce transcription
the genes are transcribed until they are turned off, or repressed
A change in the base sequence of DNA. A change in DNA will change the mRNA transcribed. When this altered mRNA is translated into protein, the incorrect base may cause the insertion of an incorrect amino acid in the protein
Sometimes mistakes are made during DNA replication
The chances of mutation can increase when cells are exposed to radiation or certain chemicals
Ex: UV light
What happens to DNA when exposed to UV light?
When DNA is exposed to UV light, any adjacent thymines in the DNA molecule may covalently bond together forming thymine dimers. Excessive UV exposure causes large numbers of thymine dimers to form in skin cells. Unrepaired damage may cause proteins that regulate the cell cycle to be made incorrectly, and this may eventually lead to skin cancer
3 ways genetic information is shared between two bacterial cells or between a virus and a bacterial cell
so-called naked DNA in solution is taken up by a bacterial cell
requires direct contact between two living cells. A sex pilus connects two cells allowing the transfer of DNA
bacterial DNA is transferred from a donor cell to a bacteriophage
recipient cell inside a virus that infects bacteria
In the three processes of sharing genetic info between bacteria, if the donor DNA is integrated into the recipient's DNA, the resultant cell is called a recombinant
In DNA, the synthesis happens from the _____ to the _____ end
Why do we have three phosphate groups in DNA?
because these are high energy bonds and we need energy to power this reaction
In DNA, the phosphate groups are attached to which end?
the 5 carbon end
In DNA, what is on the 3 carbon end?
an OH hydroxyl group
In a polymer of nucleotides, how are the carbons counted?
The chimney is the 5 carbon, and then you count 1 through 4 on each corner going clockwise to the right.
When we are synthesizing RNA or DNA, where do we add the nucleotide?
We always synthesize (in RNA or DNA) by adding energy charged nucleotide onto the 3 prime end of the growing chain. We start at 5 carbon end (at chimney) and add the nucleotides to the 3 carbon end (where the OH is). The chain grows this way.
What catalyzes DNA or RNA synthesis?
DNA polymerase catalyzes this
Where does DNA polymerase get its energy to catalyze synthesis?
Gets energy from breaking off phosphates
Helicase unwinds DNA and forms a _________
in DNA replication, at the origin of replication, the two strands of DNA separate, serving as templates for making new strands. This separation forms a replication bubble
the replication bubble grows in two directions (above and below), forming to replication forks (one to the left and one to the right)
one new strand in the replication fork that is built continuously
the 3 prime strand
the other new strand in the replication fork that is on the bottom that lags behind and is built in pieces
the 5 prime strand
relieves the strength of the twisting forces once helicase has separated the DNA strands
one piece of replicated DNA on the lagging strand
builds a new strand of DNA on either the leading or lagging strand by adding DNA nucleotides one at a time
the nucleotides on the new DNA strand must base pair with their complimentary base on the template strand (A-T, G-C)
What does DNA polymerase need before it can begin building a new strand of DNA?
A primer to tell it where to start. DNA adds a primer made out of RNA synthesized by RNA primase. Just one on leading strand. Many on lagging strand that are removed by another form of DNA polymerase after copying and replaced with DNA.
joins the Okazaki fragments on the lagging strand together
portion of the genome that contains an origin and is replicated as a unit
nucleoside triphosphate (NTP)
A molecule containing a nucleoside bound to three phosphates. Nucleotide derivatives are necessary for life, as they are building blocks of nucleic acids and have thousands of other roles in cell metabolism and regulation. NTPs generally provide energy and phosphate group for phosphorylations.
Used by RNA polymerase
deoxynucleoside triphosphate (dNTP)
The nucleoside triphosphates containing deoxyribose. They're the building blocks for DNA (they lose two of the phosphate groups in the process of incorporation)
Used by DNA polymerase
What is a key thing to remember about transcription?
The chemistry of this, the key thing to remember about transcription is that we don’t need to synthesize a copy of all of our DNA. We only need one small region.
Lets say we want to synthesize a flagella. That flagella gene is going to be one small region of the DNA. There are lots of genes in the DNA. So we only want to make a copy of that one region.
Tells pRNA polymerase where in the DNA to use to start transcription.
Sigma factor binds to a region of the DNA called the promotor.
Promoter sequence is “upstream” from the start point for transcription
a protein needed for initiation of RNA synthesis. tells it where to start and what direction to point in
sense strand or template strand
in transcription, only one strand of DNA acts as a template
nonsense strand or non template strand
The strand not transcribed
Once it attaches to the promotor via the sigma factor, where does RNA polymerase start copying from?
Everyone before the +1, all the promoter sequence is still part of the gene. We consider this to be upstream (all those in negative numbers).
tells RNA polymerase where to stop transcribing
a sequence of nucleotides in the DNA that, when transcribed, permits two complementary regions of the RNA strand to base pair, forming a hairpin loop. For reasons not yet understood this causes the polymerase to stall and dissociate from the DNA template.
the transcribing of DNA to RNA from the 5 to 3 prime direction, with a small transcription bubble
What happens when polymerase encounters the terminator?
When polymerase encounters the Terminator code it dissociates from DNA and releases mRNA
RNA polymerase goes through it, it twists and pops and forms a hairpin loop and pops the RNA polymerase off.
Ribosomes are made up of what two parts?
1. small subunit
2. large subunit
Codons are what three things?
1. unambiguous - each codon = amino acid
2. redundant - multiple codons for most amino acids
3. universal - to all organisms
specify amino acid starting point
UAA, UAG, UGA
Polymerase Chain Reaction (PCR)
is used to make multiple copies of a desired piece of DNA enzymatically
How do we get our primer to run a PCR?
From sequences on the outer edges of the gene
4 steps of PCR
1. Start with solution containing template DNA, synthesized primers, and an abundant supply of the 4 dNTPs
2. Denature double helix target DNA with heat
3. Anneal to primers specific for target DNA
4. DNA Polymerase uses dNTPs to synthesize copies of target DNA starting at the primer
Why doesn't heat denature our Polymerase enzyme?
Thermus aquaticus bacteria (Taq Polymerase)
we purify the DNA polymerase from this bacteria and we heat it up to 95 degrees, it is perfectly fine. Holds together its shape
Why do we use PCR testing? Name 5 possible reasons.
Identify presence of:
1. Antibiotic Resistance Genes
2. Toxin genes
3. Cancer diagnostics
4. Viral Infections
-- HIV screening
5. Identify bacterial species and strains
-- Use 16s rRNA sequencing
How did we get to the three domain system?
Carl Woese wanted to find evolutionary relationships and find a gene that was common to everyone but wouldn’t change too quickly. He decided to look at RRNA genes that form ribosomal RNA involved in Parkinson's. Ribosomes are different in bacteria and in euk cells and that difference is conveyed in the genes.
This revolutionized phylogenetics and it is how we got to the three domain system
What does a change in gene do to the bacteria?
It alters its genotype. This can have a profound impact on bacteria because they are haploid. Because of this, a change in genotype can easily alter the observable characteristics of an organism, its phenotype.
How do we generate diversity and variety in bacteria who are all haploid?
changes in as little as one nucleotide (one base in the DNA). Those point mutations will change the sequence of bases we see in MRNA when we transcribe the gene and may or may not result in a change in the end product in the primary sequence of our protein.
Universal code is made up of ________
What do codons do?
each codon codes for an amino acid
multiple codons that all code for the same amino acid. You can use any of the codes and get the same amino acid. So it has wiggle room.
What is the advantage of having multiple condons that code for an amino acid?
We can accommodate changes in certain spots without changing end product of amino acids.
Change in genotype (genetic sequence) results in change in protein primary structure
Changes to DNA sequence to as little as one base. If we have DNA sequence and we transcribe it to get our MRNA and we have AUG start codon so we get the MET as our sequence of amino acids that all point together to form peptide. If we change one base, we go from GCT to GTT, we can have a downstream effect, and the code on the table is changed in our sequence of amino acids. This is a missense mutation.
most common type. GCT to GCA which changes RNA synthesis which still gives us ARG because the universal code is redundant. We have a change in the gene sequence but the end product is exactly the same. Doesn’t change phenotype.
Change in Genotype results in termination of protein synthesis
One base change yields something that doesn’t code for anything so it inserts a stop codon. This ends in termination of protein synthesis. This is always a bad thing. It happened here in the third codon of the whole sequence. Not good. These are things we can see in the phenotype. If this were a flagella protein, this would stop making flagella because it would not have the protein to do so.
Change in Genotype results in change in protein primary structure
We do not change a base here, we insert or delete a base. Can happen through addition or deletion of any number of different nucleotides. Especially if you add three in a row. You have inserted an extra amino acid but that doesn't happen very often. Usually just one base added. We end up with a gobbly goup protein that doesn't do anything.
if it randomly happens
radiation or exposing it to something to mutate
Bacteria that can repair the problem if they are exposed to radiation. If they are able to repair then they are a revertant through the process of reversion. They had a mutation and then they fixed it and went back to what they had before
kill off the bacteria
Some bacteria can change what they look like on the plate
you only notice mutation under certain environmental conditions
All mutations are rare. Why?
because all cells have repair mechanisms. We can assess our genetic sequence and see if we have mismatched when synthesizing.
4 causes of mutations caused by environmental conditions
2. Alkylating agents
3. Reactive oxygen
4. Intercalating agents
What does UV do that causes mutations?
Causes covalent bonding between adjacent thymine bases forming thymine dimers which distorts DNA
Name 3 reactive oxygen species and how do they cause mutations?
They start interacting with the ends of the nucleotides that they like to form the hydrogen bonds it interacts with those and cleaves them off and does crazy chemical reactions with them and when Dna polymerase comes along it doesn’t know what they are because they have changed.
Converts amino group to a keto group
Changes cytosine to uracil
Uracil binds to adenine while cytosine binds to guanine
How do the alkylating agents cause mutations?
Largest group of chemical mutagens
Alters hydrogen bonding of bases
Nitrosoguanine is common alkylating agent
E.g. Specific mispairings occur when mutagen changes base’s structure and pairing characteristics
How do intercalating agents cause mutations? What is an example?
They are planar molecules that are flat and bind to DNA and inserts itself in between the bases. Is an intercalating agent. When it does this, it distorts how the bases all stack up on themselves and it makes it look like there is a space where a base should go. So DNA polymerase adds a base where you did not have one before in the middle of your strand. They are carcinogens because you start making all these changes to the DNA and you can have cells growing out of control.
The Ames Test
Based on observation that most carcinogens are also mutagens
Microbes used to test potential carcinogenic activity
Tests are based on mutagenic effect chemical has on microbial DNA
Ames test common chemical carcinogen test
Tests rate of reversion of Salmonella auxotroph
Also tests potential lethality
They do it backwards. We start with a strain of salmonella bacteria that has a mutation in it. And that salmonella has a mutation in one of the genes that is required for this bacteria to be able to synthesize the amino acid. Mutation in histidine synthesis. We then add our mutation causing probable chemical (mutagen) to this and mix it together and then we plate them out on plates without the histidine. And we want to know how many colonies grow. The only way they colonies grow is if a reversion mutation occurs. And that happens in the mutated gene that was mutated already. So the bacteria that are mutated can grow and synthesizes histidine. The more of these you have, then your chemical component that you added to it is probably the reason why.
3 methods of repair for mutations
1. photoreactivation (light repair)
2. excision repair (dark repair)
3. DNA polymerase
Enzyme uses visible light to break covalent bonds between bases
Thymines separated by photochemical reaction catalyzed by photolyase
Endonuclease excises damaged section
New section replicated and joined to original strand
Called UVR genes because they were studied with UV. These genes recognize again a distortion in the physical molecule. DNA strands do not line up nice and smoothly and they notice that and cut out a region of those bases to excise them. Once we cut those out we can have FNA polymerase come and add those correct bases back in. correct damage caused by reactive oxygen species damage and thiamine dimers, damaged bases, etc. the question when you have these distortions, is if you are the excision repair system, how do you know which side to replace as both sides are usually funky and do not line up right when this has to happen. Excision repair does not look at the bases, it looks at the distortion. This works with DNA methylation system which labels our DNA strands so we know which one is older and which one was most used as a template.
How does DNA polymerase execute mutation repair?
DNA polymerase is able to spot check as it adds one base at a time to make sure the new base fits in the proper orientation. If we add the wrong base, a G instead of a T, they don’t form the proper hydrogen bonds and it distorts the nice smooth two strands winding up perfectly in terms of sized and that is what DNA polymerase is able to sense that we are able to line up these bases so that the width is the same. If not lined up properly it wont be the same length. DNA polymerase can cut out a wrong area and try again and this reduces the rate of mutation by like 1000 fold. Doesn’t catch the environmental exposures where thiamine dimers form due to UV radiation where DNA is not able to tell what it is supposed to match up against. It solves some problems but not all problems.
series of enzymes that add methyl groups to the backbone all the time in the cell. So over time we accumulate a number of methyl group tabs along the length of the DNA.
A way for the molecular commands inside the cell to recognize the old template strand.
How does DNA methylation help with excision repair?
Excision repair doesn’t know which is the wrong base and which is the right base but it does know which is the old strand and the newly synthesized one based on methylation. So the default is that the old strand is the one that is right. So we replace bands on the new side when we have to with the ones that we have on the old as opposed to the ones on the new.
What catalyzes DNA methylation
separate thymine dimers using energy from visible light
recognize where dimers formed and catalyze separating bonds back apart to detach those to form thymines in normal structure. Energy requiring process and photolyase gets the energy from the sun itself.
Last ditch effort to bypass damage
Used to repair excessive damage to large sections of DNA that halts replication, leaving many gaps
Produces new DNA polymerase
Highly error prone
Mutations can arise from synthesis with new enzyme
SOS evolved to help the bacteria survive no matter what and the bacteria who come out of this are not as robust. They are missing really important genes. They become mutants like the ones we talked about that cannot synthesize their own histidine.
Vertical gene transfer
Clonal process of vertical reproduction. When we go from one parent cell to two daughter cells, those two daughter cells are directly identical. No shuffling of genes. No swapping parts. One copy of everything and we are not interacting with another cell in reproduction they divide themselves in half.
Horizontal gene transfer
Bacteria can transfer genes between each other as adults. If you could go and swap genes with other people in a room that would be horizontal gene transfer.
Three Methods of Gene Transfer
1. Transduction (not covered this exam)
taking up free floating DNA from environment.
bacteria can transform and change themselves.
example was the mice with the rough and smooth cells. there was something in the bacteria that the bacteria were taking from the dead cells and giving them the information to turn themselves into smooth cells that would kill the mice.
direct contact between two living cells.
bacterial sex. Some bacteria have genes for a structure called a pili. Uses this to connect into another cell and can send copies of DNA that way.
Is mediated by a plasmid
Only form of gene exchange in which donor survives
Two events always occur in gene exchange
1. Donor DNA is transferred and accepted by the recipient cell
2. Donor DNA is integrated on to the recipient cell’s chromosome or plasmid retained
Process of integrating DNA where your new DNA and old DNA (free standing chromosome) have sequences that are really the same in terms of the series of bases and when they are the same, there is a protein the RecA Recombinase that takes these two sequences, lines them up, and swaps those sequences. Sometimes swaps them so that they are bigger. If we are able to line up some of the ends in a chromosome then we can have the chromosome with a new DNA insert and the chromosome becomes bigger. Tied up the ends and make it a part of the giant circle.
Some bacteria, not all, that have the aility to take free floating DNA from other dead cells and incorporate them into their genome.
Competence is a condition in which bacterial cells are capable of taking up and integrating larger fragments of DNA
Competence occurs during the late log, early stationary phase
establishes a physical bridge in between two cells through which we can transfer genetic information
F Plasmid Transfer
We start with a cell that makes a pili and one that does not. The one that has the pili and has F plasmid as an F + cell. That is our donor cell and the recipient one is F- cell. Pili forms a physical bridge in between these two cells that penetrates the cell wall. We transfer DNA from along pili from cytoplasm of one cell into the other cytoplasm of other cell. We make copy of F plasmid and transfer copy to other end and at the end of the day when the pili breaks apart we end up with donor cell and recipient cell that now has extra genetic F plasmid info and can make a pili itself so it can go and contact another cell and transfer itself. A copying method.
Rolling Circle Replication
Cut one of the strands of DNA and unwinds DNA from double helix and as they unwind DNA polymerase fills in the gap behind it and as we unwind and pull off the single stranded length of DNA that can then be fed down the pili. Like a roll of paper towels. A new roll, that one part glued on is nicking and then we pull the strand off the roll but we use the cardboard roll as a template to make new copies.
is a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently. Most commonly found as small circular, double-stranded DNA molecules in bacteria
high frequency recombination
Conjugation between Hfr and F- cell
Bacterial chromosome is being transferred
Breakage of pilus usually occurs before transfer complete
only a portion of the F factor replicated and transferred.
However, portion of donor chromosome is also transferred
F- has new information but may not have the complete F factor
F- remains F-
Briefly explain the differences between F+, F-, Hfr, and F' cells.
An F+ cell will contain a circular plasmid seperate from the chromosome. the Hfr cell has the f factor integrated into its chromosome. in F' strains the f factor exists as a seperate circular plasmid but the plasmid carries bacterial genes that were originally part of the bacterial chromosome. The F- strain does not contain the f factor and can recieve DNA from cells that contain the F factor. (F+, Hfr, and F' )