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Single base substitution, causes the replacement of a single base nucleotide. This term also includes insertions or deletions
code for a stop, which can truncate the protein
code for a different amino acid.
Most proteins can withstand one or two point mutations before their function changes
Diff nucleotide but same amino acid
Also sometimes called a synonymous change, possible bc 64 codons specify only 20 amino acids
the substitutuion of a purine for a pyrimidine or vice versa
Can be drastic bc it changes the structure dramatically
changes a purine nucleotide to another purine
DNA contains what 4 Nitrogen bases?
Which are purines?
A, C, G, T
- Adenine and Guanine
- Cytosine, Thymine and Uracil
Each nucleotide contains:
A sugar, a phosphate and a nitrogen base. (Either a purine or pyrimidine)
The sugar is iehter a Ribose sugar (in RNA) or a deoxyribose sugar (in DNA)
Mice- IIIS killed and injected with IIR (non-virulent) and the mice died. Also, these bacteria retained thier type IIIS characteristics through several generations.
Avery, MacLeod and McCarty
Experiment-provided compelling evidence that the transforming principle (and genetic info) reside in DNA. They keat-kill IIIS (virulent) strain->treate each of the 3 samples with enxymes that destroy proteins, DNA or RNA (Protease, RNase, or DNase). Then add eaxch of these samples to a culture of IIR bacteria. The culture treated with DNase was the only one out of the three to not contain the transformed IIIS bacteria.
DNA consists of a large number of linked, repeating units, called ________, each containing_________.
DNA is a very long molecule sometimes termed a __________
nucleotides, sugar, phosphate and a nitrogen base
used radioactive isotopes to follow the proteins and DNA separately. DNA contains Phosphorus, so they used P-32 to follow the protein. Protein contains Sulfur but not Phosphorus, so they used S-35 to follow the protein. They grew a large batch of E.coli in a medium of either S-35 or P-32, then infected each T2 phage. Then they infected the E.coli iwth these T2 phage. Many of the phages progeny emitted radioactivity from P-32.
Watson and Crick
Discovered the 3-D structure of DNA-using x-ray diffraction and showed it as 2 strands of nucleotides wound around each other to form a right handed helix with sugars and phosphates on the outside with bases on the interior.
DNA primary structure-
refers to its nucleotide structure and how the nucleotides are joined together. Like the ladder
DNA secondary structure-
refers to the stable 3-D helix. The 2 polynucleotide strands run in opposite directions.
DNA tertiary structure-
the complex packing arrangements of double stranded DNA in chromosomes.
The additional oxygen atom in the RNA nucleotide makes it_______than DNA
more reactive and less chemically stable
This is one reason DNA is better suited as a long term repositor of genetic info.
The phosphate group in a nucleotide is a Phosphorus atom bonded to_______
- 4 Oxygen atoms. Frequently carries a negative charge which makes DNA acidic. The phospate group is always bonded to the 5' Carbon atom of the sugar
A series of nucleotides linked by phosphodiester linkages (strong covalent bonds between the 5' phosphate group of one nucleotide to the 3' Carbon atom of the next).
the backbone of the strand is composed of alternating sugars and phosphates.
The negative charges of the phosphate groups are frequently neutralized by the association of positive charges on __________
proteins, metals or other molecules.
Why are DNA strands complementary?
They are anti-parallel, and A pairs with T through 2 H bonds; C pairs with G through 3 H bonds (therefore G-C pairing is stronger)
meaning it has a right handed or clockwise spiral. This spiraling of nucleotide strands creates major and minor grooves in the helix. These features are important for the binding of some proteins that regulate the expression of genetic info.
the process of DNA expressing its genetic instruction by 1st transferrin its info to an RNA molecule. The info remains in the language of nucleic acids.
the RNA molecule then transfers the genetic info to a protein by specifying its amino acid sequence. (the info is translated, from the language of nucleotides into the language of amino acids)
What are the 3 major pathways of info flow in the cell?
- 1. Replication- info passes from DNA to another DNA molecule
- 2. Transcription- DNA or RNA
- 3. Translation- info passes from RNA to protein
creating the exact same thing
same language but different format
What is mRNA?
it's a copy of one gene made from a DNA template (through transcription or RNA synthesis), it's a template for making protein
transfers RNA - helps asseble the protein by reading (or attaching to) the mRNA, brings the amino acid to the mRNA
Each amino acid is attached to a tRNA. the old gets ejected and the new deposits another amino acid and all continues until a stop codon is encountered. You now have a polypeptide.
single base substitution, but the resulting amino acids are the same
to copy and deliver information outside the nucleus of the cell.
What does Helicase do?
'unzips DNA', breaks H bonds
how is DNA read and synthesized from _____end to _____end.
It's read from 3 to 5, and synthesizes complement from 5 to 3.
Central dogma is what 3 things?
Replication, transcription and translation
Recessive Epistasis vs Dominant Epistasis
both block a hypostatic gene.
in Rec., bb blocks A or a, but in Dom. Bb or BB blocks A or a.
- So : if A is the hypostatic, B is epistatic (in this case blocking the expression of color)
- Rec: Dom:
- 9: A_B_ Black 12:
- 3: aaB_ brown
- 4: 3: A_bb yellow 3:
- 1: aabb yellow 1:
Avery, McLeod, McCarty:
- Griffith=like the dog=animals=mice
- Avery, McLeod, McCarty= 3ppl, 3 test tubes
What do the bases look like. label the 3' and 5' end and know direction of strand
5' end has the phosphate group and 3' has the hydroxyl group
DNA replication overview
The Watson Crick model of DNA structure implies a simple mechanism for replication involving a series of enzymes: the DNA unwinds and separates (with the help of helicase), RNA primers are laid down (primase), the primers are elongated (DNA polymerase) and individual pieces are linked together with DNA ligase. Although the enzymes are slightly different, the mechanism is very much the same in both prokaryotes and eukaryotes.
4 Stages of Replication:
- 1. Initiation
- 2. Unwinding (followed by priming)
- 3. Elongation - in both priming and elongation, bases are added according to the base pairing rules previously established, always main a double stranded molecule that is anti-parallel.
- 4. Termination
1st Stage of Replication: Initiation
origin of replication -> mechanism is the smae for E.coli, cows, humans, etc.
unwinding during replication:
Helicase unwinds, SSBPs (single strand binding proteins) keep DNA from re-annealing, and gyrase reduces supercoil ahead of the fork (reduces torsional strain) to allow for continued unwinding.
draw all parts of dna replication
Polymerases in Prokaryotes
Catalyze phosphodiester bond between 3'OH and 5'P04
- direction is always 5' to 3'
- Can only add to existing 3' end, cannot initiate de novo
Primase synthesizes short stretches of RNA to provide a 3'OH for DNA polymerase to add on to.
It's only job is at the beginning of synthesis, lays down a short stretch of RNA. Many don't have proofreading step though.
3' to 5' exonuclease activity=
backspacing or proofreading
DNA pol 1
DNA pol 2
DNA pol 3
pol 1- slow, abundant, not processive enough (falls off soon, after processing only up to 200) Pol 1 replaces the RNA nucleotides of the primer with DNA nucleotides. ( This is where the nicks are left behind in the backbone, and DNA ligase seals this nick with a phosphodiester bond between the 5'P group of the initial nucleotide added by DNA pol 3 and the 3'OH group of the final nucleotide added by pol 1)
pol 2-used in repair?
pol 3- highest processivity , DNA is synthesized by this polymerase.
Enzymes in order for DNA replication
- Helicase, then Gyrase
- then Primase, then pol 3.
discontinuous DNA synthesis on the lagging strand during replication
Leading strand vs lagging strand in dna replication
leading strand is being made 5' to 3' in the same direction as the movement of the replication fork. only needs a single primer.
lagging strand is also being made in the 5' to 3' but in the opposite direction as the movement of the replication fork. series of primers required creating "okazaki fragments"
Termination of synthesis during DNA replication.
Termination can occur in different ways.
E.coli- termination protein called Tus binds to specific sequences (called ter)
others- meeting of replication forks
DNA polymerase 1
replaces RNA primer with DNA
Differences in replication in Eukaryotes (3 major differences)
- -multiple origins of replication "replicons" (due to greater size of genome)
- -more types of DNA polymerases
- -nucleosome assembly following DNA replication
yeast origins? ARS=autonomously replicating sequences
Replication in Eukaryotes overview:
Each chromosome contains numerous origins. At each origin, the DNA unwinds, producing a replication bubble. Synthesis takes place on both strands at each end of the bubble as the replication forks proceed outward. Eventually the forks of adjacent bubbles run into each other and the segments of DNA fuse...producing to identical linear DNA molecules.
Problems with multiple origins
the entire genome must be replicated ONLY ONCE in each cell cycle
- ANS: 2 distinct steps in initiation
- -origins are "licensed" to replicate in G1 with a replication licensing factor
- -initiator proteins cause the separation of DNA strands
Polymerases in Eukaryotes
at least 13 DNA polymerases
-pol alpha = primase activity and begins DNA synthesis, no 3' to 5' exonuclease
-pol gamma? = completes replication on lagging strands, has 3' to 5' exonuclease
-pol beta = DNA repair
Ends of chromosomes, are special repetitive DNA sequences (humans = TTAGGG repeated 250-1500 times)
- Telomerase extends the DNA, filling in the gap due to the removal of the RNA primer.
- -protein + RNA (CCCUAA)
- -RNA serves as template to extend 3' end
- -NOT active in somatic cells
- -active in bone marrow, stem cells, germline cells
In linear DNA with multiple origins of replication, elongation of DNA in adjacent replicons provides a 3'OH group for replacement of each primer. Primers at the ends of chromosomes cannot be replaced, because....
there is no adjacent 3'OH to which DNA nucleotides can be attached. When the primer at the end of a chromosome is removed, there is no 3'OH group to which DNA nucleotides can be attached, producing a gap.
What is semiconservative replication?
the original two strands of the double helix serve as templates for new strands of DNA. When replication is complete, two double stranded DNA molecules will be present. Each will consist of one original template strand and one newly synthesized strand that is complementary to the template.
In transcription, RNA polymerase moves ________
downstream, progressively adding nucleotides to the RNA molecule according to the sequences on the template.
Rho-independent terminator contains....
an inverted repeat followed by a Poly-A region. When the inverted repeat has been transcribed, it forms an RNA hairpin, which causes RNA Polymerase to pause. The stretch of AU base pairs is unstable and causes the RNA strand to separate from the template during the pause.
The rho-independent terminator is typically encoded in the genome and is positioned downstream from the stop codon. There is definitely a difference in structure between the two terminators; rho-independent terminators contain an inverted and repeated sequence which is able to form a hairpin structure. The sequence within the loop in the rho-independent terminator is partly species specific.
Prokaryotic DNA transcription: elongation
The elongation phase of transcription refers to the process through which nucleotides are added to the growing RNA chain. As the RNA polymerase moves down the DNA template strand, the open complex bubble moves also. The bubble is of a fixed number of nucleotides, meaning that at the leading end of the bubble the DNA helix is being unwound, while at its trailing end the single strands are being rejoined. Whereas separation of the DNA helix is permanent in replication, it is only temporary in transcription. depicts the beginning steps in transcription up to elongation and the relative positions of the bubble and the polymerase holoenzyme.Figure %: Steps in TranscriptionAs the figure shows, within the open complex bubble the DNA and RNA form a hybrid or joint complex. The exact length of this region is unknown, but it is thought to be between 3 and 12 base pairs long and is found at the growing 3' end of the RNA. The figure also illustrates how the 5' tail end of the RNA chain is separate from, as opposed to base paired to, the DNA template strand. This is another difference between DNA replication and DNA transcription; in replication, the newly synthesized DNA strand remains bound in a helix to the strand with which it has base paired. After the initial stretch of approximately 8 base pairs has been synthesized, the sigma unit, which is responsible for recognition and binding to the promoter region, is released. The core enzyme is left to polymerize the growing RNA chain alone. This leads to the continuous extrusion of the 5' end of the RNA from the enzyme complex. At normal room temperature, the rate of transcription in prokaryotes is 40 nucleotides per second.
RNA synthesis will continue along the DNA template strand until the polymerase encounters a signal that tells it to stop, or terminate, transcription. In prokaryotes, this signal can take two forms, rho-independent and rho-dependent.
The rho-dependent terminator received its name because it is dependent on a specific protein called a rho factor. The rho factor is thought to bind to the end of the RNA chain and slide along the strand towards the open complex bubble. When the factor catches the polymerase, it causes the termination of transcription. The mechanism of this termination is unclear, but the rho factor could in some way pull the polymerase complex off of the DNA strand.
The rho-independent terminator is the more simple of the two systems and as a result is also called simple termination. The rho-independent signal is found on the DNA template strand and consists of a region that contains a section that is then repeated a few base pairs away in the inverted sequence.Figure %: Rho-Independent TerminatorAs is shown in the figure, the patch is followed by a short string of adenines. When this stretch is transcribed into an RNA sequence, the RNA can fold back and base pair with itself forming a hairpin loop.As you can see, the string of adenines in the DNA sequence are transcribed into uracils in the RNA sequence. Because the uracil bases will only pair weakly with the adenines, the RNA chain can easily be released from the DNA template, terminating transcription.
holoenzyme is what two units together?
the core enzyme and the sigma subunit (the two main segments of the RNA polymerase molecule)
is responsible for carrying out the polymerization or synthesis of RNA
DNA replication: four stages:
- Unwinding (followed by priming)
- Initiation: Origin of replication, bidirectional startpoint
- Unwinding: after initiator proteins, helicase unwinds (binding to the Lagging strand template), SSBP's keep DNA from re-annealing, and gyrase reduces supercoil ahead of the fork.
- polymerases in prokaryotes: catalyze phosphodiester bond between 3'OH and 5'PO4. Direction is always 5'to3', cannot initiate de novo. can only add to existing 3' end.
- Primase synthesizes short stretches of RNA because they can start de novo.
3' to 5'
exonuclease activity = backspace or proofreading.
DNA pol 1 exonuclease activity
DNA pol 3 exonuclease activity
3' to 5' AND 5' to 3'
3' to 5' (no backing up)
Describe the function of the 8
proteins (dnaA, helicase, SSBP, gyrase, primase, DNA pol I, DNA pol III, ligase)
dnaA- binds oriC to initiate replication
SSBP-prevents unwound strands from reannealing
- creates nicks in DNA to prevent supercoiling
- RNA primers for DNA pol III to add to
- pol I-removes RNA primers, replaces with DNA
DNA pol III-main synthesis polymerase
Ligase-seal okazaki fragments together
Compare and contrast DNA polymerase I from DNA pol III (at least 2 similarities and 2 differences)
Are these in prokaryotic or eukaryotic cells? How are the DNA polymerases of the
other type of cell denoted?
- a)Both are found in prokaryotic DNA replication, have 3’ to 5’ exonuclease activity. DNA pol I is used for
- replacing RNA primers with DNA while DNA pol III is used for actual
- polymerization. DNA pol I also has 5’ to 3’ exonuclease activity, while DNA pol III does
b)Prokaryotic... Eukaryotic DNA polymerases would be denoted with greek letters (ex. DNA pol α, DNA pol δ, etc)