Unit 6 - Gene Expression/Regulation Flashcards
Explain the conclusion drawn from Griffith’s transformation experiment.
Pathogenic bacteria are killed by heat and mixed with living nonpathogenic bacteria → leads to pathogenic living cells
Discovered transforming substance = DNA (with mice)
Explain the conclusion drawn from Avery, McCarty, and MacLeod’s transformation experiment.
Tested for the transforming factor with three separate vials - DNA, proteins, RNA
Supported the claim that transforming substance = DNA
Explain the meaning of semiconservative.
DNA replication is semiconservative because each of the daughter molecules produces have one parent strand and one newly synthesized strand
Describe the steps of DNA replication.
- Begins at sites called origins of replication that have short sequences of DNA that initiation proteins recognize and attach to
- These proteins then separate the two strands and open up a replication bubble. These bubbles are spaced out on the strand
- Helicase untwists the double helix at replication forks (Y shaped regions at the end of each bubble) by breaking hydrogen bonds between the two strands
- Untwisting causes strain and tighter twisting ahead of the replication fork. Topoisomerase relieves this by breaking, swiveling, and rejoining DNA strands
- DNA polymerase comes to add nucleotides for the synthesis of a new DNA strand. However, it can only add to existing strands, not create a new one from scratch
- Primase creates a short complementary RNA chain called a primer, using the parental DNA strand as a template
- Now DNA replication proceeds in both directions along the strand. DNA polymerase can only add to the 3’ end of a primer, elongating the DNA in a 5’ –> 3’ direction.
For the leading strand, replication is continuous, into the replication fork, and only one primer is required
For the lagging strand, which is antiparallel, replication works in segments in the direction away from the replication fork. These segments are called Okazaki fragments - each of them must have a separate primer. DNA polymerase adds nucleotides to connect the primers. At the end, the RNA nucleotides from the primers are replaced by DNA nucleotides
- Finally, DNA ligase joins the sugar phosphate backbones of all the Okazaki fragments into a continuous DNA strand
What 3 factors contribute to the accuracy of DNA replication?
- Specificity of base pairing (A=T, C=G)
- Mismatch repair - special repair enzymes fix incorrectly paired nucleotides
- Nucleotide excision repair - incorrectly placed nucleotides are excised/removed by enzymes called nucleases, and the gap left over is filled in with the correct nucleotides
What are telomeres and why are they useful?
The fact that DNA polymerase can only add nucleotides to the 3’ end of a molecule means that it cannot replicate the 5’ end of the DNA molecule at the end of the chromosome. As a result, repeated rounds of replication (EX: of chromosomes for mitosis) produce shorter and shorter DNA molecules with uneven ends.
In order to prevent essential genes from being lost, the linear ends of eukaryotic chromosomes are capped with telomeres - short, repetitive nucleotide sequences that don’t contain genes. Telomeres get eroded in place of essential genes.
Euchromatin vs Heterochromatin
Euchromatin is DNA that is uncoiled and loose (non methylated and histones are acetylated) so it’s available for transcription [found in interphase cells]
Heterochromatin is DNA that is fully condensed into coils so it’s inaccessible to transcription and thus generally does not undergo it [EX: Barr bodies - permanently condensed X chromosomes in female cells]
What is the difference between replication, transcription, and translation in terms of the starting material and the product?
Replication: DNA –> DNA
Transcription: DNA –> RNA
Translation: RNA –> protein
What is the difference between replication, transcription, RNA processing, and translation in terms of location?
Replication: Nucleus in eukaryotic cells (during S phase of the cell cycle)
Transcription: Nucleus in eukaryotic cells
RNA processing: Nucleus in eukaryotic cells –> exits nucleus through nuclear pores in the membrane
Translation: Cytoplasm at ribosomes
What is the difference in gene expression between prokaryotes and eukaryotes?
In prokaryotic cells, mRNA does not undergo RNA processing and transcription & translation can occur simultaneously. In eukaryotic cells, transcription yields pre-RNA or primary transcript that undergoes processing to yield the final mRNA
Central Dogma
DNA –> RNA –> Protein
Describe the steps of transcription.
- RNA polymerase recognizes and binds to the promoter.
In eukaryotic cells, transcription factors recognize the TATA box and bind first, then help RNA polymerase bind in the correct position/orientation. The transcription initiation complex consists of transcription factors + RNA polymerase + promoter
- RNA polymerase untwists the two strands of DNA and attaches complementary RNA nucleotides to the template strand. It can only add nucleotides to the 3’ end so RNA elongates in the 5’ –> 3’ direction
- The new RNA molecule peels away from its DNA template and the double helix forms again
- After RNA polymerase transcribes a terminator sequence in the DNA (which signals the end of transcription), the RNA transcript is released and polymerase detaches. In eukaryotes, a polyadenylation signal is transcribed
Template/Noncoding strand
Provides the template for transcription; the mRNA produced is complementary to this strand
Coding strand
The sequence is identical to the mRNA produced only with T instead of U
Describe what occurs during RNA processing.
Addition of a 5’ cap (modified guanine) and a poly-A tail to the 3’ end (string of adenines). These facilitate the export of mRNA from the nucleus and protect it from degradation by hydrolytic enzymes. In the cytoplasm, they also help ribosomes attach to the 5’ end of the mRNA to begin translation
The length of the poly A tail also determines the lifespan of the mRNA fragment
RNA splicing - introns (noncoding segments) are removed and exons (coding segments) are joined together by a spliceosome. This forms a continuous coding sequence rather than segments
There are 25,000 genes but only 100,000 proteins in an organism. Propose an explanation.
In alternative RNA splicing, a single gene can encode more than one kind of polypeptide depending on which introns are moved. So processing can create multiple mRNA sequences which then produce multiple polypeptides and proteins
What did Hershey and Chase’s experiment discover?
They labeled DNA with radioactive phosphorous and protein with radioactive sulfur. Then they analyzed what happened when phages infected bacteria.
The radioactive sulfur did not get in while the radioactive phosphorous was found inside the bacteria
Conclusion: DNA is what allowed the virus to infiltrate and replicate its DNA.
Describe the structure of a tRNA molecule.
The protruding end at the top (3’) is the specific amino acid attachment site.
The bottom three bases make up the anticodon (3’ –> 5’) that base pairs with the complementary codon on mRNA
Describe the function of tRNA.
They transfer amino acids from the cytoplasm (taken from other compounds or from the surrounding solution) to a growing polypeptide chain in a ribosome
Describe the structure of a ribosome.
Made up of rRNA and protein
Consists of a small subunit that binds to the mRNA strand and a large subunit that has 3 binding sites for tRNA molecules (E, P, A sites)
Describe the three binding sites on the large subunit of the ribosome.
E site - where empty tRNA molecules exit
P site - holds the tRNA that carries the growing polypeptide chain
A site - holds the tRNA that carries the amino acid that will be added to the chain next
Describe the steps of translation.
- The small ribosomal subunit binds to the mRNA strand in such a way that the first codon (AUG) is placed in the proper position
- A special initiator tRNA, which carries the amino acid “Met” and has the anticodon UAC, hydrogen bonds to the first codon
- Then the large subunit attaches, allowing the tRNA to attach to the P site. The A site is now available to the tRNA that will bring the second amino acid.
Initiation factors are proteins that helped bring these components together into a complex
- The anticodon of the next tRNA molecule base pairs with the complementary mRNA codon in the A site
- The polypeptide chain on the tRNA in the P site is transferred to the amino acid on the tRNA in the A site
- The empty tRNA is then moved to the E site and released while the polypeptide tRNA in the A site is moved to the P site
mRNA kinda moves like a conveyer belt in the left direction from 3’ –> 5’
- This continues happening until the ribosome reaches a stop codon on mRNA. A release factor binds to the stop codon at the A site and promotes hydrolysis of the bond between the tRNA in the P site and the polypeptide (basically H2O is added instead of another amino acid)
- The complete polypeptide is released through the E site. The signal peptide (a sequence of amino acids at the leading end of the polypeptide chain) determines the destination of the protein by serving as a sort of luggage tag
What are the two main types of mutations?
- Point mutations (aka nucleotide pair substitution) - changes in a single nucleotide pair of a gene
- Frameshift mutations (aka nucleotide pair insertion/deletion) - changes in the reading frame or triplet grouping of the genetic message when it’s not a multiple of 3
What are the types of point mutations? What are their effects on phenotype?
- Silent mutation: the amino acid sequence does not change so there’s no effect on phenotype
EX: Due to the redundancy of the genetic code, CCG and CCA produce the same amino acid - Missense mutation: the amino acid sequence changes but it could have little effect if the mutation occurred in a region not essential to the function of the protein or if the new amino acid has similar chemical properties to the original one
- Nonsense mutation: the amino acid codon is changed to a stop codon which terminates translation prematurely, resulting in a shorter polypeptide and thus a nonfunctional protein; very large, harmful effect
What are the types of frameshift mutations? What are their effects on phenotype?
- Insertion: adding a nucleotide; shifting the reading frame to the right
- Deletion: removing a nucleotide; shifting the reading frame to the left
Both these mutations could have very disastrous effects because they affect multiple nucleotide pairs compared to substitutions which only affect one pair
What is a gene and what is its final product(s)?
A gene is a region of DNA that can be expressed to produce a final functional product - either a polypeptide OR an RNA molecule (rRNA, tRNA, mRNA)