Gene Expression Flashcards
What is a simple description of gene expression? What’s the central dogma?
DNA directs the synthesis of proteins
Central dogma: DNA → RNA → Protein
What did scientists suggest about what dictates genes in the early 1900’s?
Suggested that genes dictate phenotypes through enzymes
- Thought a disorder was caused by a lack of enzyme
- Mutations in Drosophila eye colour were due to missing or blocked enzyme activity
Explain George Beadle and Edward Tatum experiment and what they concluded from it
Neurospora (bread mould) cells were placed on petri plates with minimal nutrients
Cells were forcibly mutated
Found that some cells couldn’t make an essential nutrient → they couldn’t grow on minimal media but could grow on enriched media
Concluded that the gene that makes the nutrient was disabled so they couldn’t grow on media without it because they lacked the enzyme to make the nutrient
Conclusion → one gene to one enzyme
Reality → one gene to one phenotype
What is the non-template strand used for?
Also called the coding strand
Used to determine the amino acid sequence in the RNA
Could also tell us the sequence of the RNA strand → they’re the same except U instead of T
Which direction is translation? How is this different from transcription?
Translation → Ribosome reads mRNA in 5’ to 3’ direction
Transcription → DNA template strand is read 3’ to 5’ and the mRNA is made 5’ to 3’
Explain the main difference in gene expression between bacterial cells’ and eukaryotic cells
Bacterial cells have no nucleus, so DNA is transcribed and translated concurrently (all at once) → it all happens in the cytoplasm
Eukaryotic cells have the nuclear envelope, so DNA is transcribed in the nucleus, and then the primary transcript is exported to the cytoplasm for translation
How many different triplet codons are possible in the genetic code? How many different amino acids are possible?
61 possible triplet codons + 3 stop signals
20 amino acids
What are the start and stop codons?
Start → AUG - methionine
Stop → UAA, UGA, UAG
How are codons of an mRNA recognized?
Recognized by anticodons of tRNAs
What does the universalness of the genetic code mean? What’s an example of how we can use this?
Diverse life forms share the same genetic code → so one species can be programmed to produce proteins characteristic of a different species by introducing the DNA from one species into the other → isn’t always functional because not always processed correctly
Ex. we can express human genes in bacteria to produce gobs of medically-important proteins (ex. insulin) in bioreactors
Explain the location and function of the promoter and termination
The starting point is where the actual transcription will begin
“Upstream” (before the starting point), is the promoter, where RNA polymerase will attach and initiate transcription (RNA polymerase II transcribes mRNA)
- It’s usually the sequence TATA → TATA box - 25 bases upstream from start site
“Downstream” is the end point of transcription called termination
What is different from DNA polymerases and RNA polymerases?
RNA polymerases can start a chain from scratch, they don’t need to add the first nucleotide onto a pre-existing primer
What are transcription factors?
Proteins that bind to the DNA at specific locations, like the TATA box
They play a role in initiating transcription of all genes
Some transcription factors can bind to other transcription factors via protein-protein interactions
How do transcription factors bring in RNA polymerase II
More transcription factors bind to the DNA to bring in DNA polymerase II
- Transcription factors at the TATA box is perfect → brings RNA pol II to the right position so it can start transcription at the right spot
- The transcription factors with the RNA pol II (and other proteins) form the transcription initiation complex (means it’s ready to go) - Once RNA pol II leaves the initiation site, another transcription factor can bind and start transcribing
How is RNA transcription the same as DNA replication and how are they different?
Overall extremely similar
RNA polymerase II unwinds the DNA strands (not Helicase) and will expose 10-20 DNA nucleotides → multiple molecules of RNA pol II can do this at the same time on one gene
Template strand is read 3’ to 5’ and ribonucleotides polymerized 5’ to 3’ (same as DNA)
Different → uracil instead of threonine and ribonucleic acid instead of deoxyribonucleic acid
Explain the process of termination of transcription.
Termination occurs when RNA pol II hits a specific sequence of nucleotides that tells it to stop
- Prokaryotes → terminator
sequence
- Eukaryotes → polyadenylation
signal (AAUAAA)
RNA pol II keeps going for a bit, but an enzyme cleaves the RNA just after the signal, releasing the pre-mRNA for further processing
What is RNA processing?
The pre-mRNA undergoes processing where both ends of the transcript get altered and internal parts get spliced out (introns) and stitched together (exons)
*** Prokaryotic mRNAs don’t get processed
Which polypeptide is produced from a gene is dependent on which introns get spliced out and exons stitched together
What is complementary DNA? (cDNA) and what is Genomic DNA (gDNA)
Used in research
Processed transcribed RNA is converted back to DNA
Includes all the exons spliced together (coding DNA) without the introns (shorter than DNA)
gDNA is the same thing but with the introns still intact
What is the purpose of the 5’ cap and the poly-A tail in transcript processing
5’ cap is added which protest the 5’ end and helps in translation → it’s a modified guanine nucleotide
The poly-A tail is 25 to hundreds of A’s in a row that are added to the other end
This gives mRNAs stability in the cytosol and helps in translation
Explain the role of the spliceosome in RNA splicing. What is spliceosome activity an example of?
Spliceosome is an enzyme complex that binds to several short sequences alon an intron, which includes key sequences at either end
The intron is then cut by the spliceosome and rapidly degraded
The spliceosome then stitches (ligates) the exons together
- Spliceosome activity is an example of ribozymes → RNA molecules with enzymatic functions → disproves the idea that all biological catalysts are proteins
What is alternative RNA splicing?
What does this result in?
The process by which different introns and exons are spliced out within a single transcript to yield different polypeptides
This results in the number of proteins that can be made being higher than the number of genes present within the genome
What are proteins’ domains?
Functional regions where stuff happens (ex. Active sites for catalytic reactions, or allowing for the protein to integrate into the plasma membrane)
What is exon shuffling?
Introns can facilitate crossing over of the exons of an allele during meiosis
Non-homologous mixing and matching of exons between different genes
Could lead to novel combinations of amino acids→ novel proteins → novel functions
Where does protein translation occur
the cytosol
What components are needed for translation?
- mature mRNA exported from nucleus
- tRNAs that recognize the codons on the mRNA with their anticodons on the tRNA
- each tRNA (with a specific anticodon) has the proper amino acid for the codon covalently attached to it
- anticodons are written 3’ -> 5’ to align with the 5’ -> 3’ codons
- Ribosomes (made from rRNA and proteins)
Role of tRNAs
Transfer amino acids from the cytoplasm to the growing polypeptide chain in the ribosome
Explain the structure of tRNA
each is a relatively small RNA that is twisted into a ‘cloverleaf’ shape (2D shape) with some intra-molecule base pairing (hydrogen bonding) - like an “L” shape in 3D
many bases in each specific tRNA are chemically modified
What is amino-acyl tRNA
tRNA + the proper amino acid attached
how many codons are there and how many amino acids?
61 codons + 3 stop signals
20 amino acids
tRNAS are matched with their correct amino acid with an _________
Aminoacyl-tRNA synthetase
ex. tyrosyl-tRNA synthetase can only recognize the tRNAs that have anicodons that matches the Tyr codons and can only recognize the amino acid tyrosine (like a puzzle shape and the amino acid and tRNA are the puzzle pieces coming together)
_________ provides the energy required for wynthetase to form a covalent bond between the tyr-tRNA and tyrosine
ATP hydrolysis
When synthetase cataylyzes the covalent bonding of the amino acid to its specific tRNA, the tyr-tRNA is now __________
charged, and can be used by the ribosome during translation
What is a wobble?
The flexibility in the third position in the triplet of anticodons
Explain the structure of ribosomes
composed of:
- rRNA
- proteins
2 subunits (large and small)
Synthesized in the nucleus, then exported to the cytoplasm
fully formed when mRNA is threaded through
What are the three tRNA ‘sites’ in the large ribosomal subunit?
A = aminoacyl (where charged tRNAs attach)
P = peptidyl (the tRNA holding the polypeptid is)
E = exit (where ‘spent’ tRNAs are ejected
Functions of ribosomes:
Facilitate codon-anticodon matching (on binding sites)
Form peptide bonds
rRNAs bind an mRNA at the _____ end and finds the first AUG codon
5’
What are the 3 stages of translation
Initiation
Elongation
Termination
Describe the steps in the Initiation stage of translation- where does small ribosomal subunit attach?
A small ribosomal subunit binds to 5’ end of mRNA and looks for the first AUG codon
The first tRNA carrying Met (“start”) codon attaches by codon/anticodon binding
After GTP is hydrolyzed, the large ribosomal subunit attaches
This positions the first tRNA (with Met) to the “P” site
The ribosome then starts “reading” the rest of the mRNA codons in the 5’ to 3’ direction
Explain the steps of elongation in translation
The anticodon of the next aminoacyl tRNA pairs with the complementary mRNA codon in the A site
- GTP is hydrolyzed to help with accuracy and efficiency
An rRNA molecule in the large subunit catalyses the formation of the peptide bond between the amino group of the new amino acid in the A site and the C-terminus of the amino group in the P site
The polypeptide on the P site is removed and put onto the amino acid in the A site
Another GTP is hydrolysed - the ribosome translocates the tRNA in the A site to the P site. The empty tRNA from the P site moves to the E site
The A site is now empty and can receive the next tRNA
Explain what happens during the termination stage in translocation- what recognizes the stop codons?
A stop codon enters the A site
- A release factor is a protein (not tRNA) that recognizes stop codons by amino acid / base interactions
The release factor causes the ribozyme to hydrolyze the polypeptide from the tRNA in the P site
The ribosomal subunits dissociate
In post-translation modifications, ________ determines the primary structure, and the primary structure determines ________
Genes , Shape
What are the different options for post-translation modifications?
Folding in based on the amino acid sequence
Some amino acids may be chemically modified by attaching sugars, lipids, phosphate groups, etc.
Enzymes may remove one or more amino acids from the leading (amino, N) end
Enzymes may also cleave the polypeptide into smaller chains or join two ore more polypeptides into a single chain
What are the two kinds of ribosomes?
Free Ribosomes are in the cytosol and synthesis polypeptides that will stay in the cytosol
Bound ribosomes are attached to the cytosolic side of the rough ER/nuclear envelope
- make proteins for the
endomembrane system
Polypeptides destined for the endomembrane system (or secretion) are marked by a _________
Single peptide
- 20 amino acids that are recognized by a signal-recognition particle (SRP)
What does the SRP (signal-recognition particle) do
Escorts the ribosome to a receptor protein in the ER membrane, locks it on, and polypeptide synthesis continues there
What makes a polyribosome
Once a ribosome has synthesized a polypeptide from a portion of mRNA, another ribosome can attach and synthesize another of the same polypeptide. - like beads on a string
How is a polyribosome made in Eukaryotes
mRNAs are circularized before they’re translated
As the first ribosome moves away from the start codon, another ribosome can attach and begin translation
the PolyA tail shortens over time. If it gets too short the mRNA can no longer be circularized and will no longer be translated
- polyA tail acts as a ‘timer’ - mRNAs with longer polyA tails are translated for longer, yielding more protein product per mRNA
How are polyribosomes made in Porkaryotes
No nucleus, so transription and translation happen at the same time - no mRNA processing
As soon as RNA polymerase starts making an mRNA, ribosomes attach and start making the encoded polypeptide
As in eukaryotes, this rapidly produces lots of polypeptides from a single mRNA molecule
What is a point mutation
Change in a single nucleotide
What is a nucleotide-pair substitution?
Replacement of a nucleotide and its partner with another nucleotide pair –> can have silent, missense, or nonsense
What occurs from a silent, missense, and nonsense mutation?
Silent = no observable effect on phenotype since the amino acid didn’t change
Missense = one amino acid is changed for another
Nonsense = a point change results in a stop codon, resulting in termination
What can result from a nucleotide-pair insertion or deletion?
May alter the reading frame and result in a frameshift mutation if the insertions/deletions are not in multiples of three
Results in extensive missense mutations since the codons will code for a different amino acid before finally ended at a nonsense mutation
How do mutations arise
Mutagens –> chemical or physical agents that alter DNA
What are the two ways for E.coli to regulate tryptophan production?
The cells reduce the activity of the enzymes that make tryptophan (faster response)
Inhibit the expression of the genes that encode the tryptophan enzymes (slower response)
What are operons - how are they regulated?
A group of genes with a common function
Environmentally regulated by a common promoter region that includes an operator region (“on-off switch”) that helps regulate whether the genes are expressed
Regulated by regulatory genes (ex. trpR) elsewhere in the genome - they are either activator (on) or repressor (off) proteins that regulate whether the operon will be transcribed
How do repressor proteins work? How are they encoded?
the repressor protein binds to the operator, thus preventing RNA polymerase from binding to the promoter to initiate gene transcription
- repressor proteins are specific for the operator within a specific operon - encoded by a regulatory gene, which sits upstream from the operon. --> its an allosteric protein because it's constantly expressed and produced in its inactive form, but it only works when tryptophan (or a diff aa) binds to it
What would happen if the tryptophan level is too high and expression needs to be turned off?
Tryptophan binds to the repressor protein, activating it (acts as a corepressor)
the active repressor binds to the operator sequence in the trp operon promoter
there it blocks the binding of RNA polymerase, so the operon isn’t transcribed
thus, the tryptophan biosynthetic enzymes aren’t produced
The trp operon is a _______ operon because it is always on but can be inhibited when a molecule binds to it
repressible
The lactose operon is an ________ operon (off, but can be turned on) when a specific molecule binds to a regulatory protein
inducible
If E.coli doesn’t normally use lactose as an energy source and the lac operon is normally turned off, how would it be turned on?
The lac repressor protein is made in an active form and prevents the lac operon genes from being transcribed
If lactose is abundant in the environment, allolactose (an inducer) binds to the repressor and inactivates it
Thus, the genes that allow the bacteria to use lactose are transcribed
What are repressible operons and inducible operons usually associated with?
repressible -> anabolic processes
inducible -> catabolic processes
How can signal transduction pathways help the cell to sense when the environment changes and genes need to be expressed to fulfill a need? (cAMP concentration example)
cAMP binds to a cAMP receptor protein (CRP, an activator), which binds to the DNA at a specific location upstream of the lac promoter
- increases the infinity of RNA polymerase for the lac promoter, increasing transcription of the lac operon, stimulating gene expression and the production of the enzymes required to break down lactose
If the glucose concentration increases, cAMP concentration falls (due to less signal transduction), which reduces binding of CRP, which reduces the amount of RNA polymerase that binds to the operon (thus reducing transcription)
Humans have approximately ________ genes, and only about ______ of them are expressed in any specific cell type like the liver or muscle cells. What accounts for this?
20 000
20%
Housekeeping genes - expressed in most cells all the time (actin, tubulin, ATP synthase, etc.)
What are the two kinds of differential gene expression?
Spatial expression -> different cell types express a set of genes so that the cell can do its job (ex. liver cells expressing toxin degrading enzymes)
Temporal expression -> expressed at different times (ex. in response to the cell perceiving a hormone, at specific points in development, etc.)
What are the two ways for differential expression at the transcriptional level to occur?
- whether the gene is in a heterochromatin (inactive) or euchromatin (active) area of the genome. Done through chromatin modification.
- Whether specific transcription factors are present in the cell that will allow for the transcription of a specific gene
DNA is associated with proteins to form __________
chromatin
What modifications can alter how tightly the chromatin is packed?
Chemical modifications of the histone tails -> the N-terminal ends of the histone proteins that protrude from the nucleosome as tails
________ of the histone tails promotes looser chromatin structure
_________ of the tails compacts the chromatin into a heterochromatin state
These are called _______ marks
Accetylation
Methylation
Epigenetic
How do transcription factors work?
Upstream from the TATA box are control elements that are specific nucleotide sequences contained within enhancer regions
- different genes have different combinations of these elements
If specific TFs are produced in the cell (activators or repressors), they can recognize and bind the sequences of the enhancer elements
This binding either increases or decreases the rate of transcription of the gene
About _______ percent of all genes encode transcription factors
10
How do activator transcription factors speed up the rate of transcription
If an activator transcription factor binds the enhancer elements, a DNA-bending protein bends the DNA, placing enhancers + activators close to the TATA box and transcriptional start site
Here they interact with a group of proteins that form the mediator complex, which ‘mediates’ the interaction of the activators and the initiation complex
Mediator helps bring in the general transcription factors and RNA polymerase II to form the initiation complex
This increases the rate at which RNA polymerase II is recruited to the transcriptional start site, so the rate of transcription increases
What are transcription factor master switches?
Some transcription factors are more ‘upstream’ in a developmental pathway than others because they can regulate the transcription of other transcription factors
The master switch will turn on other TFs which will all turn on more TFs
What are frameshift mutations
Insertions or deletions that change the reading frame
What are tumor suppressor genes and what is a common example of one?
They are genes that encode proteins to stop cell division if something is wrong (like if the DNA is damaged)
p53 is a transcription factor that produces a protein that inhibits the cell cycle if DNA is damaged - it also produces DNA repair enzymes (however it’s often mutated in cancer cells, which means they would continue dividing even with the damaged DNA)
ALL cancers exhibit __________
unregulated cell division
