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Topics 2.6, 2.7, 7.1-7.3 (DNA Replication, Transcription, and Translation), Molecular Biochemistry, Molecular Genetics, Gene Regulation, and Mutations, DNA Sequencing, Molecular Biology Final
Terms in this set (66)
Describe 3 differences between DNA and RNA.
Note: Deoxyribose has one less oxygen molecule than ribose
Describe the types of bonds involved in DNA structuring.
Covalent bonds between nucleotides.
Hydrogen bonds between base pairs.
Explain the use of Taq DNA polymerase during the PCR.
After DNA is heated to break hydrogen bonds and DNA primers added when cooled to prevent the re-annealing of the parent strands, Taq DNA (which can operate in higher temperatures) rapidly replicates the DNA.
Describe the role of RNA primase during transcription.
RNA primase unwinds DNA's double helix and moves along the ANTISENSE strand, pairing complementary RNA bases.
Describe the difference between the antisense strand and sense strand.
The antisense strand is what serves as a template for RNA creation through transcription. Therefore, the un-transcribed sense strand has the desired genetic coding which needs to be sent out of the nucleus.
What is the start codon of all translation processes?
AUG, which codes of methionine
Structure of nucleosomes. GO!
8 histone proteins. 2 copies of 4 types.
Name and describe the proteins involved in DNA replication.
Helicase: breaks H bonds between bases to separate DNA strand
Topoisomerase: Enzyme which lays ahead of replication fork
Single-Stranded Proteins: Binds to parental strands to keep them form re-annealing, allowing replication to properly occur.
DNA Primase: Creates RNA primer to start DNA replication
DNA Polymerase III: Creates new DNA strand with complementary base pairing in the 5'-3' direction
DNA Polymerase I: Replaces RNA primer with DNA sequence
DNA Ligase: Closes Okazaki fragments caused by replication on the lagging strand running opposite of replication fork
Explain how gene expression can be regulated by proteins.
Enhancers: Proteins can bind to enhancer sequences of DNA to trigger the start or increase of transcription of the desired genes.
Silencers: Sequences of nucleotides which slow the rate of transcription when proteins bind to them
Promoter-Proximal Elements: See Enhancers. Only difference is that these elements are closer to the actual promoter of transcription
Explain the role of nucleosomes during transcription regulation.
When the tail of a nucleosome undergoes methylation (addition of a methyl group), it tightens up and slows/prevents transcription. The opposite occurs during acetylation.
Describe the process which creates mature mRNA.
Pre-mRNA has its non-coding introns removed by sn RNPs (Snurps), and a 5' cap and poly-A tail is added to the strand.
Explain how mRNA splicing leads to the regulation of gene expression.
mRNA can be spliced in a multitude of ways as certain exons can also be removed with the introns. For example, the protein tropomyosin can be coded without the 2 exon (skeletal muscle) or the 3 and 10 exons (smooth muscle)
Explain the role of tRNA-activating enzymes.
tRNA-activating enzymes serve as the location at which tRNA binds to its respective amino acid. This is made possible with ATP being transformed into AMP.
Outline the process of translation.
1. Initiation: mRNA binds to small ribosomal subunit and initiator tRNA anti-codon binds to codon of mRNA.
2. Elongation: Large ribosomal subunit attaches to small unit, placing initiator tRNA into the P-site. A second tRNA attaches to the A-site and a peptide bond forms between the two amino acids. The ribosome then translocates in the 5'-3' direction, sending the P-site tRNA to the E (exit) site, allowing another tRNA to bind to the A-site.
3. Termination: The ribosome reads the stop codon, preventing another tRNA from binding to the A-site. The polypeptide is then freed and the ribosomal unit disassembles.
Explain the purpose of non-coding DNA.
- Production of tRNA and rRNA
- Enhancer and silencer sequences
- Moderately and highly repetitive sequences (i.e. telomeres)
DNA loops around histones to form the nucleosome "beads on a string." H1 binds to linker DNA and nucleosome to stabilize.
Highly condensed, transcriptionally inactive.
Barr bodies (X chromosomes) are an example.
Less condensed, transcriptionally active.
"euchromatin is expressed"
Template strand is methylated in DNA replication, which allows mismatch repair enzymes to distinguish between new and old strands.
DNA methylation at CpG islands represses transcription
Reversibly represses DNA transcription
relaxes DNA coiling, which allows transcription
makes an RNA primer to which DNA Pol3 can initiate replication
RNA dependent DNA polymerase that adds DNA to 3' end to prevent loss of genetic material.
*often dysregulated in cancer cells, allowing unlimited replication.
Missense DNA mutation
replacement of a base that leads to substitution of a single amino acid
-Sickle cell disease = substitution of glutamic acid with valine
Nonsense DNA mutation
replacement of a base that leads to termination of translation (UAG, UAA, UGA)
Frameshift DNA mutation
Insertion or deletion of a base that affects that entire downstream sequence
-Duchenne muscular dystrophy and Tay-Sachs disease
Site where negative regulators bind
noncoding regions of DNA that influence the activation of genes;located at distance upstream from the genes they exert control over.; bring certain transcription factors into contact with promoter regions and act enrich transcription
common element in eukaryotic cells is TATA box
A-T rich regions of DNA involved in positioning the start of transcription
proteins that exert transcriptional control over the genome; some bind to enhancer sequences and others to promoter sequences; help RNA polymerase find and bind to given promoter region
untwists the double helix at the replication fork
helps relieve the strain caused by DNA untwisting. It breaks, swivels and rejoins DNA strands
starts a complementary RNA chain using parental DNA strand as a template
the fragments made by the lagging stand; short segment of DNA
joins Okazaki fragments together
the DNA strand synthesized continuously during DNA replication;
ALWAYS 5' TO 3' DIRECTION
the DNA strand synthesized discontinuously during DNA replication;
ALWAYS 5' TO 3' DIRECTION
The bonds linking "A" to "T" are formed because the atoms have polarity. What does "polarity" mean and why is the oxygen atom polar?
Polarity means that the electrostatic charge is asymmetrically distributed about the atom. Or one side of the atom has a higher local concentration of electrons than the other. Oxygen is polar because it has two lone pair electron oribitals oriented away from the position of the double bond. Electron distribution.
If the molecule "A:T" were floating freely in solution would the interaction between "A" and "T" be stable? Why or why not.
* No it would not be stable because in aqeuos solution the bond between A:T will have to compete with water which is "sticky" and polar. Also this bond is relatively weak, especially in comparison to water.
*No, there are only two H bonds linking A to T which are weak bonds because they are competing with H bonds of similair strength to water molecules which are abundant in solution. The kinetic motion of water molecules can break A:T H bonds and replace those bonds with bonds to water molecules.
DNA oxidation. DNA oxidation is the process of oxidative damage on Deoxyribonucleic Acid. It occurs most readily at guanine residues due to the high oxidation potential of this base relative to cytosine, thymine, and adenine.
Describe the role of 8-oxogunanine DNA glycosylase in reducing the problem associated with guanine oxidation
* 8-oxoguanine DNA glycosylase is one of the repair mechanisms for DNA
*It recognizes 8-oxoguanine, a damaged/modified base in DNA and catylizes the excision of of that base from the ribose subunit. Without this mechanism the prescence of 8-oxoguanine would result in a mutation in the DNA because 8-oxoguanine would pair with A instead of C
DNA polymerase is a proofreading enzyme. At the molecular level how does it identify mistakes that it has made?
The active site has a shape that fits proper base pair combos. Improper base pairs have a shape/structure that collides with the side walls of DNA polymerase active site. The side wall collision activates triggers the '3-5' exonuclease activity.
Describe how telomerase activity results in multiple short DNA sequence repeats at the ends of chromosomes
Precise base pairing between the RNA molecule and the telomere DNA results in the placement of a specific part of the RNA as a template for DNA synthesis. The newly synthesized DNA has a sequence that base pairs with the telomerase RNA in the same way that it did in the previous cycle. As a result the next round of DNA synthesis produces the same nucleotide sequence. The cycle can occur multiple times, resulting in DNA sequence repeats.
Why is DNA replication left incomplete when the replication fork gets to the end of the chromosome?
The lagging strand is left incomplete because it has nowhere to put a primer upstream of the last nucleotides in the DNA strand. Thus there is no way for DNA polymerase to add complementary nucleotides to these parts of the DNA.
*Because it needs a RNA template to finish the ends
What role does the RNA molecule play in the activity of telomerase?
It helps telomerase bind to the end of the chromosome by base pairing with the nucleotide sequence at the end of the DNA . It also serves as the template strand for the addition of new telomerase DNA
One of the first steps of the Illimina sequencing method is to break the genomic DNA into small fragnments (approx 500 base pairs in size). Why is this step necassary?
The PCR like step where fluorescent DNA bases are added occurs at the ends of the sample DNA fragments. Becuause the adapter sequences for binding DNA to plate and serving as prime binding sites for polymerase attatch to the ends of the DNA fragments in the sample. Unbroken DNA has fewer ends and the middle sections are too far from the ends to be sequenced
How does RNA polymerase determine which nucleotide (A,U, C or G) to add to an RNA sequence during transcription? Where is this determination being made in the diagram above?
RNA determines which nucleotide to add by adding the complementing nucleotide from DNA to the RNA strand. The determination is being as the DNA strand is read
What is an enhancer element and how does it relate to the control of transcription?
It is a DNA sequence that serves as a binding site for transcriptional regulators which control the recruitment of RNA polymerase to the promoter
What is the +1 site of a gene?
*It is the first nucleotide read by RNA polymerase during transcription.
*It is where RNA polymerase is going to transcribe first, found with the promoter sequence.
How do transcriptional activators and repressors identify specific binding sites on the DNA?
Amino acids in the proteins make electrostatic bonds to the DNA . Contact is made with the sides of the nucleotide bases. Often in the major grooves which provides more polar groups and therefore more nucleotide specific information compared to the minor groove. Contact with bases are needed for sequence specificity.
How can a transcriptoin factor that binds 5,000 base pairs away form the +1 site have the effect of activation gene expression?
DNA is flexible so it can bend and loop to help gene expression in this way. Transcription factors can also bend and loop and also communicate with promoters and the mediator complex.
Suppose a transcriptional activator named Trx1 has the effect of turning on the expression of multiple other genes. What DNA sequences might the activated genes have in common.
Enhancer elements that contain DNA sequences that are specifically recognized by Trx1. Core Promoter sequences introns/exons/splice site junctions etc.
What is the function of tRNA synthetases?
They charge tRNA's with amino acids. They have binding specificity that ensures that tRNA's are charged to the correct amino acid. The fidelity of the match is important for accurate protein synthesis.
The active site of isoleucyl tRNA synthetase has two separate pockets for binding amino acids, as shown. Why is this the case and how do these binding pockets work together in isoleucyl tRNA synthetase function?
The first pocket is the active site and permits charging with I and V. The second pocket is a proofreading site that only fits V. In this case, valine is cleaved away and the tRNA can now be charged again with I or V. If I is inserted is does not fit into the active site, and therefore remains charged to the tRNA.
In humans, DNA polymerase works at a rate of 50 nucleotides per second. How the incoming nucleotides triphosphates brought to the DNA polymerase catalytic site so quickly?
The rate of movement of small molecules like nucleotides in the cytoplasm is extremely fast. There is no mechanism directing nucleotides to DNA pol, and nucleotides move about the cytoplasm in random directions. However, the rapid movement of molecules in the cytoplasm means that collision occurs between DNA polymerase and nucleotides very frequently. Much greater than 50 nucleotides per second.
Why do nucleotide triphosphates move around in solution and why is their rate of movement fast copared to much larger enzymes like Uracil DNA glycosylase and DNA ligase?
*Nucleotide triphosphates move around in solution because of water which is polar and competitive. They are faster because of their size and don't have as much mass.
How does the cell recognize the unusual characteristics of the viral genome and act against it?
The cell recognizes the double stranded RNA viruses, double stranded DNA viruses, and single stranded RNA viruses
How can foreign (viral) double stranded RNA be cleaved and used against itself?
The DICER/RISC complex
What is PKR signalling in relation to defense against viruses?
Through PKR signalling, a key factor in translation initiation is deactivated by phosphorolation. When EIF2 is phosphorolated the Met tRNA + small ribosomal subunit fail to sit down on the start codon causing an inhibition of protein synthesis. This also causes death of the cell.
How do cells respond to viral infection?
1. Inhibit all gene expression and drop dead (EIF2 and PKR)
2. Express defensive proteins and signaling factors.
(Now the alarm can spread to neighboring cells and the immune system, which will also express protective proteins. RIG-1 activation induces defensive gene expression.
What is APOBEC?
A cytosine deaminase and cytosine is changed to uracil.
What does APOBEC do to the virus?
It causes hypermutation of the viral DNA. When the virus infects a new cell, the APOBEC that they carry deaminates new DNA made during reverse transcription. The new viral DNA is hypermutated and likely non-functional.
How does splicing machinery recognize the boundaries between introns and exons?
snRNP's include small RNA's with nucleotide sequences that base pair with mRNA sequences that serve as splice site junctions. snRNP's are a component of splicing machinery.
What is the virus's defense against PKR?
K3L is a protein expressed from the poxvirus genome. It is a structural mimic of EIF2 and protects the virus by competitive inhibition. During infection the virus will flood the cell with enough K3L to allow some EIF2 to remain active (un-phosphorolated). As a result the cell's translation machinery doesnt shut down and the virus parts continue to be made.
What is the virus's defence against the RISC complex?
Viral protein p19 binds to any short dsRNA sequence. During infection, this will include any fragments of the viral genome. P19 binding to RNA blocks assembly into RISC complex
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