Genetics final
True or false: During transcription, the RNA molecule is synthesized in the 3' to 5' direction
FALSE
True or false: Transcription of a given gene typically takes place on only one of the two DNA strands
TRUE
True or false: During transcription, the RNA molecule that's synthesized is antiparallel and complementary to the template DNA strand
TRUE
True or false: During transcription, the RNA molecule that is synthesized will have U in place of T
TRUE
Three "rules" of genetic material
Must replicate faithfully: the process of copying DNA is highly accurate, genetic instructions are passed on new cells with no errors)
Must encode the phenotype: carry instructions to produce the organism’s observable traits (in charge of all the physical characteristics)
Must have the capacity to vary: DNA must be able to change overtime/create genetic diversity
History of DNA
90k years ago, human bones of a female hybrid (made of two different human breeds) were found in a cave
Problems with isolating and sequencing DNA
Damaged DNA, problems isolating it
The discovery of the structure of DNA was important because
you cannot understand genes without knowing about DNA, and that leads to understanding which genes encode for which proteins
Chemists that studied DNA roles: Miescher
Chromatin (through the study of white blood cells that have large nuclei, isolated it and discovered that chromatin was the substance inside the nucleus, which is made up of DNA and protein)
Chemists that studied DNA roles: Kossel
DNA contains four nitrogenous bases (and named them adenine, thymine, guanine, cytosine)
Chemists that studied DNA roles: Levene
nucleotide-simple structure, less variation (sugar, phosphate base, nitrogenous base), identified that nucleotides are the units of nucleic acid, incorrectly thought that there was a definite arrangement of nucleotides
Chemists that studied DNA roles: Chargaff’s rules:
DNA varies in base pair composition, definite ratio in different nitrogenous bases. Discovered that adenine is always = to thymine, and guanine is always equal to cytosine (Chargaff’s rules)
If C = 20%, then G = 20%, adding to 40%. This would mean that T = 30% and A = 30% to add up to 100%.
What is the point of the "three experiments"?
Supporting all genetic info is encoded in the structure of DNA or RNA
What is the transforming principle
Something in the virulent bacteria mixed with something in the rough type (non virulent bacteria) and ended up killing a mouse. The rough type bacteria was transformed.
Experiment 1: Griffiths Experiment
Most important: tested two different strains of bacteria, smooth strain (IIIS) = virulent, and rough strain (IIS) = non-virulent. They then heated up the virulent (IIIS) strain which didn’t kill the mouse when injected (due to killing the enzymes). Then, they injected the heat killed (IIIS) strain with the non-virulent (IIS) strain and found that it killed the mouse.
Second experiment: Avery Macleod & McCarty’s experiment
revealed the transforming principle through the tests using RNase, protease, and DNase.
RNase was there to destroy any RNA in the sample
Protease to destroy any proteins in the sample
DNase cleaves any DNA.
Samples with the RNase and protease injected resulted in the transformed bacteria, but the sample with DNase injected resulted in the transformation not taking place.
DNA was the cause of transformation
Third experiment: Hershey Chase
Bacteriophage, a virus that infects bacteria (ex. It infects e. coli).
They radioactively labeled protein and DNA to track where they went.
They labeled protein with sulfur, and DNA with phosphorus, letting them sit in two jars with the bacteriophage present.
They blended and spun them and found that the bacteria did not have sulfur present, but had phosphorus present.
DNA is present in the virus, not protein. Therefore genetic material is DNA not protein
They couldn’t use radioactive carbon because it’s present in both DNA and protein
Watson and Crick major breakthrough
DNA was the source of genetic info
They did not conduct any experiments themselves, just used already available information
What is DNA and its structure
DNA: primary structure is deoxyribose is in DNA, double helix
Each nucleotide contains: a deoxyribose sugar, phosphate group, one of the four bases (adenine, guanine, thymine, cytosine)
Stores genetic info long term
What is RNA and its structure
ribose is in RNA, single stranded
Each nucleotide contains ribose sugar, phosphate group, one of the four bases (adenine, guanine, uracil, cytosine)
Carries instructions from DNA to build proteins
Purines
Double ring bases 6 and 5 carbon ring: Guanine, Adenine.
PO4- group at 5’ position, OH group at 3’ position.
Phosphodiester bonds linking adjacent nucleotides created from 5’ PO4- + 3’ OH, all covalent bonds
Pyrimidines
Single ring bases: Cytosine, Thymine, Uracil. At the 5’ position there is phosphorus attached to it, covalent bonds
Two strands of DNA in a double helix are held together by
Hydrogen bonds between complementary bases on antiparallel strands.
Base bonding: The OH end is considered the
3’ end
Base bonding: the phosphate group is considered a
5' end
Nucleotides are linked together with a
phosphodiester bond
How many bonds between G - C
3 (triple) bonds (more stable, therefore more resistant to heat)
How many bonds between A - T
2 bonds, less stable
In RNA how many bonds between A - U and G - C
A - U = 2 bonds
G - C = 3 bonds
Hybridization
Occurs between single stranded nucleic acids as long as they share a region that can complementary base-pair and they are antiparallel
DNA, A model
Right-handed helix with less H2O present and shorter and wider
Unlikely to physiologically exist
DNA, B model
Right-handed helix with lots of H2O present and most stable physiologically
Most predominant form in cell
DNA, Z model
Left-handed helix with zigzag sugar phosphate backbone
Stretches of DNA sequences (GCGCGC)
Associated with transcriptionally active regions of DNA
How is DNA packed into cells
DNA appears in a clump called the nucleoid
Supercoiled DNA is overwound or underwound, causing it to twist on itself
Positive supercoil vs negative supercoil
Positive supercoil is when the DNA is overrotated, the helix twists on itself, negative supercoiling is when the DNA is underrotated, the helix twists on itself in the other direction
100bp = 10 complete rotations in relaxed dna
Example: if there are 300 bp and 20 rotations, it would be negatively supercoiled
Chromatin structure
Made up of DNA + protein
Present in euchromatin or heterochromatin
Can be changed through methylation, when a methyl group is added to the DNA, affecting phenotypic expression of the gene
Epigenetic affect
methylation, when a methyl group is added to the DNA, affecting phenotypic expression of the gene
Mice test: more methylation, more different colour
Euchromatin
Arms of the chromosome
Crossing over happens more often
Heterochromatin
Centromere
Nucleosome
Consists of eight histone proteins which the DNA wraps 1.65 times
Attached to each other by the Linker DNA
Proteins
Made up of chains of amino acids, have primary, secondary, and tertiary structure
Act as enzymes, build and repair tissues, transport oxygen
Not able to replicate like DNA
Replication
Doubling of DNA
Has to be incredibly accurate, one error per million base pairs would lead to 6000 mistakes every time a cell divides
Conservative replication model
All of the DNA from the parent is intact, and another new set of DNA is produced in the first doubling. In the second doubling, the identical parent strand conserves itself and makes another strand whereas the new strand makes identical copies of itself (25% conserved, 75% new)
Dispersive replication model
The DNA breaks into segments and each segment serves as a template for new DNA to build on it
Semiconservative replication model
Half of each strand is the parent one, other half is the new one
Meselson and Stahl’s Experiment
They used two isotopes of Nitrogen (N14, more common, and N15, more rare)
Centrifuged both of them to see if they settled at the bottom or not
N15 is heavier, so it settled at the bottom when DNA was added
Daughter strands were neither N14 or N15 and settled in the middle
After the 2nd replication, one strand was lighter and one was heavier
Semi conservable model
DNA is replicated by what model
Semi-conservative
Replicon
units of replication, segment of DNA where replication happens, 200,000-300,000bp in length
Replication origin
the particular base pair where replication begins on the segment (replicon)
Theta replication
Circular DNA, takes place in E. coli
Where two replication forks move in different directions
Replication fork
place where the unwinding of DNA takes place
DNA replication takes place
unidirectionally as well as bidirectionally (mostly bidirectional in e.coli)
What is the function of DNA ligase?
connects Okazaki fragments by sealing nicks in the sugar–phosphate backbone
Which of the following eukaryotic DNA polymerases and their role are INCORRECTLY paired?
a. (delta); lagging strand synthesis of nuclear DNA
b. (epsilon); translesion DNA synthesis
c. (eta); translesion DNA synthesis
d. (gamma); replication and repair of mitochondrial DNA
e. (theta); DNA repair
b. (epsilon); translesion DNA synthesis
Which of the following molecules is synthesized using nucleotides containing the bases adenine, guanine, cytosine, and uracil?
a. RNA only
b. DNA only
c. both RNA and DNA
d. neither RNA nor DNA
a. RNA only
Which of the following statements is TRUE regarding transcription in most organisms?
a. All genes are transcribed from the same strand of DNA.
b. Both DNA strands are used to transcribe a single gene.
c. Different genes may be transcribed from different strands of DNA.
d. The DNA template strand is used to encode double-stranded RNA.
e. The DNA nontemplate strand is used to encode single-stranded RNA.
c. Different genes may be transcribed from different strands of DNA.
Which of the following is observed in prokaryotes but not in eukaryotes?
a. UGG is an example of a stop codon only found in prokaryotes.
b. An mRNA can be translated by only one ribosome at a time in prokaryotes.
c. The 5' end of a prokaryotic mRNA can be translated while the 3' end is still being transcribed.
d. Translation does not require any protein factors in prokaryotes.
e. In prokaryotes, ribosomes move along an mRNA in the 3' to 5' direction.
c. The 5' end of a prokaryotic mRNA can be translated while the 3' end is still being transcribed.
Which of the following statements describes the “wobble” rules CORRECTLY?
a. There is a flexible pairing between tRNA and amino acid as there are more tRNAs than the number of amino acids.
b. The number of the genetic code exceeds the number of amino acids available in the cell.
c. There are multiple tRNAs that may bind to the same amino acids.
d. There are multiple codons that may code for the same amino acids.
e. The third base pairing between the tRNA and mRNA is relaxed.
e. The third base pairing between the tRNA and mRNA is relaxed.
Which of the following mRNA codons will bind to the tRNA anticodon 5 GCU 3, considering wobble–base pairing rules.
a. 5' AGU 3' and 5' AGC 3'
b. 5' UGA 3' and 5' CGA 3'
c. 5' AGC 3'
d. 5' CGA 3'
e. 5' AGU 3', 5' AGC 3', 5' AGA 3', and 5' AGG 3'
a. 5' AGU 3' and 5' AGC 3'
. Which of the following statements does NOT describe the events in prokaryotic translation elongation?
a. The nucleotides in the Shine–Dalgarno sequence of the mRNA pair with their complementary nucleotides in the 16S rRNA.
b. A ribosome with a growing peptide attached to a tRNA in the P site accepts a charged tRNA with the next amino acid into the A site. The charged tRNA enters as a complex with EF-Tu and GTP.
c. If the anticodon of the charged tRNA matches the codon, GTP is cleaved and EF-Tu exits and is regenerated to EF-Tu-GTP by EF-Ts.
d. A peptide bond is formed by the peptidyl transferase activity of the large subunit rRNA. The polypeptide chain on the tRNA in the P site is transferred to the amino acid on the tRNA in the A site.
e. The ribosome translocates toward the 3 end of the mRNA with the aid of EF-G and GTP hydrolysis. The empty tRNA that was in the P site moves to the E site and exits. The tRNA with the polypeptide that was in the A site moves to the P site.
The nucleotides in the Shine–Dalgarno sequence of the mRNA pair with their complementary nucleotides in the 16S rRNA.
9. During initiation of translation:
a. the initiator tRNAmet binds to the A site of a ribosome.
b. specific rRNA base pairs with a sequence in mRNA to position a ribosome at the start codon.
c. IF-3 must be recruited to the 30S ribosome in order for the 70S initiation complex to assemble.
d. there is no energy expenditure as the tRNA binding to mRNA is via complementary base pairing.
e. both 70S and 30S ribosome subunits must simultaneously recognize an mRNA to bind.
b. specific rRNA base pairs with a sequence in mRNA to position a ribosome at the start codon.
E. coli lac operon control by lacI is:
a. negative inducible.
b. negative repressible.
c. positive inducible.
d. positive repressible.
e. attenuation.
a. negative inducible.
Steps of Replication
1.Double stranded DNA unwinds at replication origin
2. Single stranded templates are created for synthesis of new DNA, this is where the replication bubble appears, with a replication fork at each end.
3. The forks proceed around the circle
4. Two circular DNA molecules are produced
Replication in Eukaryotic cells
Linear replication, multiple points of origin where replication bubbles appear
DNA synthesis takes place on both strands at the end of the bubble as the replication forks proceed outwards.
Eventually, the forks of adjacent bubbles run into each other resulting in the fusion of the DNA segments
This produces two identical linear DNA molecules
How many points of origin in prokaryotes?
One
Replication can only go from
5’ to 3’
Leading strand
the strand that continuously is made in the 5’ to 3’ direction towards the replication fork
Lagging strand
produces fragments
Okazaki fragments
discontinuously synthesized short DNA fragments forming the lagging strand
How is unwinding initiated
Initiator protein
DNA gyrase in unwinding
Relieves strain ahead of replication fork
DNA polymerase
Synthesizes new DNA molecules by adding nucleotides one by one
Primers
an existing group of RNA nucleotides with a 3′–OH group to which a new nucleotide can be added; they are usually 10–12 nucleotides long. – Primase: RNA polymerase
Primers are added at the beginning of every
Okazaki fragment
What carries out Elongation
DNA polymerase III
Each active replication fork requires five basic components:
1. Helicase to unwind the DNA
2. Single-strand-binding proteins to protect the single nucleotide strands and prevent secondary structures
3. DNA gyrase to remove strain ahead of the replication fork
4. Primase to synthesize primers with a 3′–OH group at the beginning of each DNA fragment
5. DNA polymerase to synthesize the leading and lagging nucleotide strands
Replication requirements
Template strand
Raw material: nucleotides (unit of DNA, made up of a sugar, phosphate, and base)
Enzymes (to catalyze reactions) and other proteins
Topoisomerase I
causes single strands to break
Topoimerase II
causes a double strand to break (gyrase)
What is DNA replication
Doubling of DNA, RNA formation caused by DNA polymerase
why does RNA polymerase carry out transcription slower than DNA polymerase
Replication is faster because replication process involves doubling the entire DNA, where the transcription process only happens at a relatively slower speed to pieces and fragments of DNA.
RNA primary and secondary structure
Primary: Straight strand, has base pairs and Uracil instead of Thymine
Has OH group, unlike DNA
Secondary: Folded structure with base pairs labelled
Ribosomal RNA: rRNA:
making of ribosomes
Messenger RNA: mRNA:
coding information transcribed from DNA
Transfer RNA: tRNA:
link between messenger and ribosome, brings amino acids to add to the polypeptide chain (how protein synthesis takes place)
Small nuclear RNAs: snRNAs:
ability to make bonds with small ribonuclear proteins
CRISPR RNA (crRNA)
is only present in prokaryotes and helps with getting rid of foreign DNA.
The RNA molecules that are involved in RNAi are
siRNA (small interfering RNA)
The RNA found in the testes is
pirRNA
Transcription
Synthesis of RNA molecule from a DNA template
Requirements for transcription
One DNA template is required, also
Ribonucleic material/bases (ribonucleotide triphosphates)
Transcription apparatus (enzymes along with supporting proteins)
RNA molecules are synthesized that are
complementary and antiparallel to the template strand
Direction of RNA synthesis is from
5’ to 3’
Upstream
Any sequence that is present before RNA coding
Downstream
Any sequence after the gene
The promoter region is not
transcribed
Difference between template and non-template strand
Template strand is being used for transcription of the gene
The non template strand is not copied
One codon is made up of
3 nucleotides
Initiation of transcription
The substrate for transcription – Ribonucleoside triphosphates—rNTPs added to the 3′–OH group of the growing RNA molecule
The sigma (σ) factor: binding to the promoter when transcription starts, the transcription will not take place if there is no sigma factor
Transcription Bubble
Since RNA synthesis doesn’t require a primer, new nucleotides are added to the 3’ end of the RNA molecule, and DNA unwinds at the front of the transcription bubble, and then rewinds