DNA lectures (Verkade) Flashcards
All of Heather Verkade's lectures
What cellular processes involve DNA?
- DNA replication
- Transcription
- DNA regulation
- (Recombination)
- Mutations
- DNA repair
What cellular processes involve RNA?
- genetic material for viruses
- translation
- enzymatic activity (in translation)
- translocation
- regulation of gene expression
What cellular processes involve nucleotides?
- DNA and RNA structure
- Energy (ATP)
- NAD+ and FAD+ (transport of electrons)
What are the three components of a nucleotide?
- Nucleotide base
- Pentose sugar
- Phosphate group
(Picture: Deoxyribose Nucleic Acid)
Can you recognise whether a nucleotide is a dNTP or and NTP from the structure?
Yes, from the presence (or a lack thereof) of an oxygen molecule (or hydroxy group) from the 2’ carbon
What is a pyrimidine and what is a purine?
Pyrimidines are single ring bases. They are thymine, cytosine and uracil. Purines are double ring bases, which are guanine and adenine
What are the base pairings found in DNA and RNA?
DNA
- Adenine - Thymine (A - T)
- Guanine - Cytosine (G - C)
RNA
- Adenine - Uracil (A - U)
- Guanine - Cytosine (G - C)
Pyrimidines and purines are different sizes, so why is DNA always the same width?
Because purines always bind with pyrimidines, maintaining an even width
General structure of a single strand of DNA with 5’ and 3’ label
Representation of a double-stranded piece of DNA. Indicate bases and their pairing and the 5’ and 3’ of BOTH strands
Which of the base pairings is stronger, and why?
G - C bonds are stronger because they have 3 hydrogen bonds, whereas A - T bonds only have 2 hydrogen bonds
What is the type of chemical bond that exists between the phosphate and the pentose of the neighbouring nucleotide?
phosphodiester bond (covalent)
What is the type of chemical bond that exists between the base pairing nucleotides?
hydrogen bonds
What does it mean that DNA is antiparallel?
That the two DNA strands run in opposite directions to each other (i.e. one runs from 5’ to 3’ and the other runs parallel to it, but from 3’ to 5’)
Why was the discovery of the structure of DNA central to our understanding of inheritance?
Because seeing the complementary nature of the double helix structure shows an ability to be replicated with only half the material (semi-conservative replication)
What is different about the tertiary structures of DNA and RNA?
DNA
- double helical strand
- twisted around in a helix
RNA
- single strand
- forms bulges, internal loops and hairpins when bound with itself or another RNA (hydrogen bonds)
Chargaff’s rules
DNA based content is different between species, the same all over one organism, and does not vary over time or environment
- The number of adenine always equalled the number of thymines
- The number of guanines always equalled the number of cytosines
Where are the 5’ and 3’ ends of the pentose sugar?
What are the 3 possible structures of DNA?
A form:
- Can be made in the lab
- But doesn’t occur naturally
- The major difference is the conformation of the deoxyribose sugar (the puckering)
- right-handed helix
B form:
- naturally occurring form of DNA
- right-handed helix
Z form:
- “may occur some time during early cell life” (or development)
- But this is only a theory
- left-handed helix
(Picture (from left to right): A form, B form and Z form DNA)
What is the difference between leading and lagging strand synthesis?
Difference between leading and lagging strand synthesis:
- leading strand synthesis is the continuous elongation of the new strand (from 5’ to 3’)
- lagging strand synthesis is made in Okazaki fragments read from 5’ to 3’ building (but blocks start at the 3’ end)
List things about the replication of the two strands of DNA that are the same. What is different?
- DNA gyrase AKA topoisomerase (to stop supercoiling)
- helicase (unwinds DNA)
- goes through DNA polymerase III
- Run through β-clamps
- Single-strand binding proteins (SSB)
- DNA polymerase I to replace primers (but once in leading vs many times in lagging)
Differences
- RNA primers (one vs. many)
- Okazaki fragments in the lagging strands
- DNA ligase - to repair gaps between Okazaki fragment in lagging strand
- Inability to complete all sequences in lagging strand
In what phase of the cell cycle does the cell replicate its DNA?
In the S (synthesis) phase
Which of the DNA polymerases are involved in DNA replication (and which is the main one)?
DNA polymerase I and DNA polymerase III. DNA polymerase III is the main one.
Which polymerase is part of a large multisubunit complex in eukaryotic DNA replication?
DNA polymerase III
DNA replication extends in a 5’ to 3’ direction. Draw out a replicating molecule of DNA (both strands, separating in a replication fork) and indicate exactly where the next nucleotide will be added.
What group is at the 3’ end of the DNA, and what group does it attach to in the incoming dNTP? What molecule is released from the reaction?
A hydroxy group. It connects to the α-phosphate group. The molecule that comes off is a pyrophosphate (two phosphate groups).
What ions are an essential part of the catalytic site in DNA polymerase, what amino acids are coordinating them, and what do the ions do?
- Mg2+ ions
- Coordinated by Aspartic acid
- And they coordinate the phosphate groups of the incoming dNTP and the O- at the 3’ end of the growing strand: this coordination allows a better position for the reaction to take place
Considering DNA polymerase I and III, which inserts more bases in a row (is more processive)? Which has 3’ to 5’ exonuclease activity? Which has 5’ to 3’ exonuclease activity? What is exonuclease activity and in carrying out what functions do the polymerases use their exonuclease activity?
- DNA polymerase III
- DNA polymerase I and III
- DNA polymerase I only
- Exonuclease activity removes nucleotides from the RNA
- 3’ to 5’: proofreading (backspace)
- 5’ to 3’: removes the primer
What is an origin of replication?
A sequence that DnaA binds to that initiates DNA replication
What is a replication bubble?
A bubble caused by DnaA-dependent denaturation (tension in the DNA in the DUE region (Rich in A-T pairs) causes the DNA to unwind)
What is a replication fork?
The replication fork is the area where the replication of DNA will actually take place
The bacterial origin of replication has an AT-rich region. What is the significance of this?
When DnaA-ATP binds to the origin of replication, the tension caused by the binding will cause DNA denaturing easier with AT bonds than GC bonds due to the fewer number of hydrogen bonds
Draw a table in which you list the role of DNA replication proteins: pol I, III, DnaA, ligase, primase, helicase, topoisomerase (gyrase), sliding clamp, clamp loader, single-stranded binding protein
Can you identify where the DNA replication proteins are in a diagram of a replication fork, including one in which the lagging strand is looped?
What is the primer in DNA replication made of and what is it for? i.e. Why can’t the strand of DNA just be replicated without a primer?
It’s made out of a small strand of RNA primer, to allow for the new nucleotide to bind
What is an Okazaki fragment? What protein removes the primer? What does DNA ligase do and why is this necessary?
Okazaki fragments are short, newly synthesized DNA fragments that are formed on the lagging template strand during DNA replication
How many molecules of DNA polymerase III are in the replication protein complex at a replication fork?
Two
Draw a DNA molecule with an open replication bubble with both replication forks. Label the directions of the strands of DNA. Can you identify the leading and lagging strands at both replication forks?
What are the mechanisms involving DNA polymerase to prevent the insertion of incorrect nucleotides? Does DNA polymerase use 5’ → 3’ exonuclease activity or 3’ → 5’ exonuclease activity to achieve this?
Two mechanisms:
- Binding of nucleotides (the shape of the active site and the hydrogen bond strength)
- Proofreading (from 3’ to 5’ direction)
Think about the chemical reaction that added the nucleotide in the first place
When a nucleotide is removed by an exonuclease process because it was incorrect, can it then be added in the next correct position? Explain why or why not.
No. A dNTP is added to an existing strand of DNA. When it is added, it loses two phosphates and essentially becomes a dNMP, which cannot be added into the DNA, because it can’t facilitate the synthesis reaction
What does it mean that DNA replication is semi-conservative?
The new strands are made from a new strand and a strand from the parent DNA double helix
What is a telomere, and what does telomerase do? What would happen if telomerase did not function in your cells? Why do prokaryotes not need telomeres?
- A telomere is a region of repetitive nucleotide sequences at each end of a chromosome, which protects the end of the chromosome from deterioration or from fusion with neighbouring chromosomes.
- Telomerase is a reverse transcriptase enzyme that carries its own RNA molecule which is used as a template when it elongates telomeres
- DNA would continue to degrade causing mutations
- Because their DNA is circular
What are some differences between DNA polymerase I and III?
- Polymerase I: only one subunit
- Polymerase III: approx. 10 subunits
- Pol I has a slower polymerisation rate
- Pol III has a higher processivity
DNA helicase is spinning motor of 4 subunits. TRUE or FALSE
FALSE
DNA helicase is a spinning motor of 6 subunits. It uses ATP to spin down the DNA in a 5’ to 3’ direction.
What is a mutation? For a child to inherit a mutation, where in the body must that mutation occur? What risks are associated with a mutation in another part of the body?
- A mutation is a change in nucleotide sequence, that is inheritable
- It needs to be in the germ cells (or sex cells)
- Cancer
What are some causes of DNA damage?
- Radiation
- UV light
- Radio and chemotherapy
- Replication errors
- Viruses
- Metabolism
- Alkylating agents
Draw a small segment of DNA with a mismatch. What is a biological process that could have caused this mismatch? What is the importance of the fact that the new strand of DNA (during DNA replication) is not methylated for a short time in E-Coli when repairing this type of mismatch? Does this same methylation difference and DNA repair mechanism work in eukaryotes?
- Biological process: DNA replication errors
- The methylation is used to identify which strand is the old (i.e. template) strand.
- Does not work, because methylation occurs at a different nucleotide and this mechanism is unique to e-coli
What is depurination? What is deamination? What sorts of repair pathways repair these?
- Depurination: removal of a purine base (guanine and adenine) from the sugar (replaced by an OH group… usually)
- Deamination: removal of an amino (NH2) group (replaced by an O… usually)
- Repaired by base excision repair
What is the purpose of a uracil glycosylase and why is it important?
It cuts out deaminated cytosine (which is uracil), so that the correct base can be put in
There are four steps in base excision repair. Name the protein that catalyses each step, and what happens.
- DNA glycosylase: Cuts out damaged base
- AP endonuclease: (with phosphodiesterase) Puts a nick in the gap, by removing the sugar phosphate
- DNA polymerase I: Replaces a few bases from 5’ to 3’ (with NTPs → Deoxyribose phosphate + dNMPs) - leaves a nick at the end
- DNA ligase: Repairs nick after the new DNA @ 3’ end
What is a thymine dimer? Why is it a problem for the DNA? What is the usual source of a thymine dimer? What method of repair is used?
- Adjacent thymines joined together with a strong C-C covalent bond
- It causes kinks in the DNA chain, which causes big disruptions in the DNA (for one, it won’t be coded)
- It is usually caused by a catalysed reaction with UV light
- It is repaired by nucleotide excision repair
There are four steps in Nucleotide excision repair. What protein catalyses each step and what happens?
- excinuclease: creates 2 nicks, one upstream of the lesion and one downstream of the lesion
- DNA helicase: unwinds the DNA strand between the two nicks
- DNA polymerase I (or DNA polymerase ε): Creates a phosphodiester bond at the nick with a 3’ hydroxyl group and adds new correct DNA bases
- DNA ligase: Repairs nick at the end of new strand of DNA
Why can’t a thymine dimer be removed by base excision repair?
Base excision repair only repairs single bases, and as the dimer is two thymine bases joined together, they’ll be pretty hard to remove
What is a sunburn?
A radiation burn caused by exposure to the UV produced by the sun (DNA damage in skin and causes inflammation)
Tans are healthy. TRUE or FALSE?
FALSE
Tans are the physiological response from DNA damage. DNA damage in skin cells triggers the production of melanin, which somewhat protects from further damage.
Why is a double stranded DNA break potentially dangerous? What can cause a double stranded DNA break? What might happen if there is a double stranded DNA break during DNA replication?
- It leaves the DNA susceptible to being chewed away - bases can get lost
- Ionising radiation, errors in DNA replication, oxidising agents, other metabolites, gamma radiation (from sun and minerals in the earth)
- Some DNA may not be coded or replication errors can occur
What kind of DNA damage is repaired by Non-Homologous End Joining, and what is the downside of this repair?
- Double stranded breaks are repaired by NHEJ
- There is an unrecoverable loss of nucleotides due to degradation from ends
- And without a sister chromatid nearby, no information can be provided to show what the missing (degraded) code information was
As you age, DNA damage is repaired, so when you are older your cells will not have any more mutations than when you are younger. TRUE or FALSE?
FALSE
As you age, you’ll accumulate small changes to DNA due to NHEJ (Non-Homologous End Joining), and also mutations that the cell wasn’t able to identify and correct before replication finishes
What are the four phases of the cell cycle? What is a checkpoint? What does it block?
- G1 phase, S phase, G2 phase, M phase
- A checkpoint is one of several points in the eukaryotic cell cycle at which the progression of a cell to the next stage in the cycle can be halted until conditions are favorable
- It stops cells from dividing under unfavourable conditions
What is apoptosis? Where does apoptosis occur naturally during development? (there are two examples in humans and one in frogs)
- Apoptosis is a form of programmed cell death, or “cellular suicide.” It is different from necrosis, in which cells die due to injury.
- Apoptosis is an orderly process in which the cell’s contents are packaged into small packets of membrane for “garbage collection” by immune cells.
- Apoptosis removes cells during development, eliminates potentially cancerous and virus-infected cells, and maintains balance in the body.
- In frogs: the cells in the tadpole tail undergo apotosis and is absorbed back into the body and recycled
- In humans: The webbed skin between our fingers undergo apotosis, and self-recognising B cells also undergo apotosis so as not to destroy cells in our own body, neural connections are also refined via apotosis
What are the hallmarks of cancer?
- Sustaining proliferative signaling
- Evading growth suppressors
- Avoiding immune destruction
- Enabling replicative immortality
- Tumor-promoting inflammation
- Activating invasion and metastasis
- Indusing angiogenesis
- Genome instability and mutation
- Resisting cell death (inc. apotosis)
- Deregulating cellular energetics
When must DNA repair occur in the cell cycle?
Before mitosis and replication, that is what the checkpoints are for, to allow the DNA time to repair
What kinds of damage can occur to a single base?
- Spontaneous oxidative damage (red)
- Hydrolytic attack (blue)
- Uncontrolled methylation (green)
(Picture: All the possible spontaneous alterations to DNA - size of arrows indicates frequency)
What is produced during transcription? What is the direction of transcription? Why are Molecular Biologists always referring to the coding strand when they talk about a gene?
- RNA equivalent of the “coding” DNA strand is produced
- 5’ to 3’ directionality
- Because coding strand is the “code” of the RNA and subsequently, it is also the code that proteins will be coded from
What is the name of the protein that carries out DNA-directed RNA synthesis?
RNA polymerase
Would a CMP, CDP or a CTP be chemically added to the mRNA during transcription? Which end is it added to? What molecule is released during the process? What type of metal ion is required for this chemical process? What amino acids coordinate these ions?
- CTP would be added to the 3’ end of the growing RNA strand
- Pyrophosphate is released during the process
- Like with DNA polymerisation, Mg2+ ions are needed and they are coordinated by Asp (D)(Aspartate/Aspartic acid)
Their names are the same as their locations
What are the names of the two sequences that define the start of a gene in E.coli?
- -35 Region
- -10 Region
Both are promoter sequences
What are the names of the three stages of transcription?
- Initiation
- Elongation
- Termination
On a prokaryotic or eukaryotic chromosome there are genes on both strands of DNA. TRUE or FALSE?
TRUE
Genes are on both strands on DNA, therefore it is really important that the gene on the correct side is produced, as they can lead to very different RNA sequences, and consequentally, very different proteins
What are the roles of the three RNA polymerases in eukaryotes? Which one is responsible for transcribing most mRNAs, and what is the main sequence that guides it to the promoter?
- RNA polymerase I: transcribes mostly ribosomal RNA (rRNA); rRNA are not translated
- RNA polymerase II: transcribes most messenger RNA (mRNAs); it is the TATA box that guides it to the promoter
- RNA polymerase III: transcribes tRNAs, some rRNAs and other RNAs
Write out a double stranded DNA, label the 5’ and 3’ ends of each strand, label the location of a gene, label the two strands that are relevant during transcription. Where are the promoter sequences? Draw RNA polymerase II and show which strand it is binding, and which direction it is going.
RNA polymerase II is a large complex of many proteins. TRUE or FALSE?
TRUE
RNA polymerase has 12 subunits
What is the difference between a general transcription factor and gene regulatory protein (transcription factor)?
- A general transcription factor is required for the expression of almost all genes
- Gene regulatory proteins (AKA transcription factors) regulate the expression of a subset of genes
What is TBP and what is its role during transcription? What is TFIIB and what does it do? What is TFIIH and what is its role? What modification occurs to the CTD of RNA Pol II to initiate transcription? What do termination factors achieve?
- TBP: TATA-binding protein, specifically recognises TATA box
- TFIIB: Transcription factor II B, binds to TBP; recruits Pol II-TFIIF complex
- TFIIH: Transcription factor II H, unwinds DNA at the promoter (helicase activity); phosphorylates Pol II (within the CTD); recruits nucleotide-excision repair proteins
- CTD is phosphorylated, which allows the polymerase to escape the promoter
- CTD is dephosphorylated by termination factors
What three types of processing occur to the initial transcript to make a mature mRNA? Where is the mRNA located during these processing events?
- 5’ cap
- splicing
- polyA tail
- Everything kinda happens at the CTD (C-terminal domain)
What is added to the 5’ end on a mRNA? What is this for?
7-Methylguanosine is added to the 5’ end of the mRNA via a 5’,5’-Triphosphate linkage
It protects the 5’ end from destruction as the mRNA gets transported out of the nucleus to the ribosomes for translation
7-methylguanosine is added to the 5’ end of an mRNA with a phosphodiester bond. TRUE or FALSE?
FALSE
7-Methylguanosine is added to the 5’ end of an mRNA with a 5’,5’-Triphosphate linkage
What does the spliceosome do? Where in the pre-mRNA are the sequences located that are recognised by this complex (be specific)? What parts of the spliceosome complex bind to and recognise these sequences?
- Spliceosomes recognise sequences in the pre-mRNA and splice them out
- The sequences are located:
- At the very beginning [GU]
- In the middle [A]
- At the end [AG]
- U1 snRNP and U2 snRNP recognise [GU] and [A] respectively and bind to them
The process of splicing regularly introduces small variations between each mRNA. TRUE or FALSE?
TRUE
Alternative splicing
The spliceosome contains both proteins and RNAs. TRUE or FALSE?
TRUE
The proteins (different colours) form a ring around the snRNAs (small nuclear RNAs)
There is a sequence at the end of the mRNA that signals for a polyA tail to be added, but this is not present in the original gene and has been added by the RNA polymerase. TRUE or FALSE?
FALSE
It’s part of the original gene (AATAAA)
What are the stages of transcription in eukaryotes and what occurs at each stage?
- Assembly and Initiation: TBP binds to TATA box; TFIIB binds to TBP and recruits the Pol II-TFIIF complex ⇒ Transcription bubble forms ⇒ CTD is phosphorylated during initiation (polymerase leaves promoter region)
- Elongation: aided by elongation factors after TFIIE and TFIIH dissociate
- Termination: Elongation factors dissociate; CTD is dephosphorylated as transcription terminates ⇒ process facilitated by termination factors
What happens to the DNA when TBP binds to the TATA box?
It causes a kink/bend in the chain
What is the CTD (carboxy-terminal domain) of the RNA polymerase II like?
- Many repeats of -Y-S-P-T-S-P-S- in a β-spiral
- It is flexible so doesn’t always stay in a spiral (changes shape depending on what it is bound to)
- linked to the rest of the protein with a linker
How is the 5’ cap attached to the end of the new RNA strand?
- The cap-synthesizing enzyme is attached to the phosphorylated CTD
- When the 5’ end of the new RNA gets to the enzyme, the cap is attached (don’t need to know mechanism)
- The new RNA strand is then tethered to the end of the CTD with CBC while elongation occurs
What are the recognisable sequences that spliceosomes recognise in introns?
- “GU” sequence
- “A” sequence
- “AG” sequence
How does the splicing mechanism work?
- U1 snRNP attaches to the [GU] sequence and U2 snRNP (with ATP) attaches to the [A] sequence
- U4-U6 and U5 also attach to the intron sequence (and become an inactive spliceosome)
- With ATP, U1 and U4 is removed and the spliceosome becomes active
- It gets the intron into the lariat formation and then releases the intron where it will be recycled into the cell
- The spliced mRNA is then ligated
How is the polyA tail formed?
- At the 3’ end of eukaryotic mRNAs there is a sequence: 5’ AAUAAA 3’
- The sequence is recognised by polyadenylation factors, which are attached to the CTD
- An endonuclease cleaves the mRNA at this site (where the mRNA is still attached to the CTD) and the mRNA leaves RNA polymerase II
- Polyadenylate polymerase comes in and adds a large tail of Adenine bases to the 3’ end of the mRNA (using ATP)
- the polyA tail determines stability of the RNA; longer polyA tail = longer mRNA life
The genetic code of humans (which codon encodes each amino acid) is almost universal. TRUE or FALSE?
TRUE
Universal for almost all organisms
What are the names of the three stages of translation?
- Activation of amino acids: the tRNA is aminoacylated
- Initiation: mRNA and aminoacylated tRNA bind to a small ribosomal subunit, then a large subunit binds as well
- Elongation: successive cycles of aminoacyl-tRNA binding and peptide bond formation occur until ribosome reaches a stop codon
- Termination: translation stops when a stop codon is encountered; mRNA and protein dissociate and ribosomal subunits are recycled
(Protein then folds)
What are the major molecules that take part in translation?
- tRNA (translates)
- Aminoacyl-tRNA synthases
- Ribosomes (made of proteins and rRNA)
- mRNA
What is a ribosome and what type of molecules is it made up of?
The ribosome is a complex molecular machine, found within all living cells, that serves as the site of biological protein synthesis (translation). It is made up of two subunits that are made of proteins and rRNA.
What two molecules bind to the mRNA to initiate translation?
- amino-acylated tRNA
- small ribosomal unit
NOTE: Large ribosomal unit only attaches to the mRNA AFTER the amino-acylated tRNA and the small ribosomal unit have attached to the mRNA
What attaches the amino acid to the tRNA? Is there one for each amino acid, or is there one that does all amino acids?
- Aminoacyl-tRNA synthetases
- There is one for each amino acid
The tRNA and the amino acid are attached with hydrogen bonds. TRUE or FALSE?
FALSE
The tRNA and the amino acid are attached with a covalent bond
What is the critical sequence in the tRNA that directs translation? What is the name of the sequence? How does it relate to the mRNA?
- Anticodon
- It is the complementary antiparallel sequence to the codons in the mRNA
Write out a double-stranded piece of DNA, label the ends and label the strands for translation. Identify the location of the promoter. Write out the mRNA that is translated from this DNA, label the ends, and write out the key sequence within the tRNA that binds to the first codon in the mRNA. Label the ends of this key tRNA sequence.
What is a silent mutation? What is a missense mutation? What is a nonsense mutation? What might a frameshift mutation cause? What is the effect on protein function of each of these?
- Silent: Nucleotide pair substitution that has no effect on the amino acid sequence
- No effect on protein function
- Missense: Nucleotide pair substitution that encodes for a different amino acid
- Can cause problems in the fold, in catalytic function, and in coordinating functions (can also cause problems in binding)
- Nonsense: Nucleotide pair substitution that produces premature termination (stop codon)
- same as missense
- Frameshift: can cause nonsense or extensive missense
- Insertion/Deletion of 3 nucleotides = addition or loss of an entire amino acid
How does a prokaryotic ribosome identify the initiation codon in an mRNA? What part of the eukaryotic mRNA is initially recognised and bound by the ribosome?
- Shine-Dalgarno sequence (ribosome binds here)
- In eukaryotes: ribosome binds at the 5’ cap
The three sites in the ribosome are E, P and A. What is each site named for?
- E: Exit site - tRNA exit from this site
- P: Peptide site - site where the tRNA that holds the peptide resides
- A: Aminoacyl-tRNA site - site where a new aminoacyl-tRNA binds
The growing polypeptide chain is moved from tRNA to tRNA as translation continues. TRUE or FALSE?
TRUE
Which end of the growing polypeptide protrudes out of the ribosome?
The amino end
When does a tRNA move from the A site to the P site? When does it move to the E site?
- When the growing polypeptide chain attaches to the A-site tRNA
- When it passes the growing polypeptide chain to the A-site tRNA
- It leaves the ribosome shortly afterwards
The polypeptide and the empty tRNAs both exit from the E site as translation continues. TRUE or FALSE?
FALSE
Empty tRNAs exit from the E-site, the polypeptide exits from a different gap in the ribosome
What needs to bind to terminate transcription? What does it recognise to bind?
- Release factors that bind to the A-site of the ribosome terminate transcription
- It recognises stop codons
- UAA
- UAG
- UGA
What are two different cellular locations in which ribosomes might be found? Why would they be in two different locations?
- Cytoplasm
- Rough Endoplasmic Reticulum (Rough ER)
- It depends on where they are needed (if they need to be modified or transported into the cell membrane or out of the cell = Rough ER)
What is a signal peptide? Give an example of a location to which a protein might need to be trafficked.
A signal peptide is near the start of the protein sequence for those proteins that are modified, trafficked and/or secreted. It signals SRP to transport them to the rough ER
If all cells have the same genes, how do they have different functions?
Gene regulation
What is an operon? Do both prokaryotes and eukaryotes have operons?
In genetics, an operon is a functioning unit of DNA containing a cluster of genes under the control of a single promoter.
Only prokaryotes have operons
How many mRNAs are produced from one operon? How many polypeptides?
- One mRNA
- and usually 2-6 polypeptides (depends on # of genes)
What is a promoter? What is an operator?
- The promoter is the region where the RNA polymerase binds to
- The operator comes after the promoter and is a repressor binding site
What is the function (overall) of the genes in the trp operon?
To create L-tryptophan
What binds to the promoter of the trp operon? What binds to the operator?
- RNA polymerase binds to the promoter of the trp operon
- The trp repressor (activated with L-tryptophan) binds to the operator
What is produced by the trpR gene? What is the function of this protein?
- Inactive trp repressor
- Binds to the operator to stop gene expression when there is enough/too much L -tryptophan
What molecule binds to the TrpR protein? Is the TrpR molecule functional with or without this molecule? (ie. does its binding activate or inactivate the TrpR protein?)
- L-tryptophan
- It activates the repressor (the repressor cannot function without L-tryptophan)
If tryptophan is present, are the trp genes expressed? Is an mRNA produced? Are proteins produced?
- No, no and no
If a mutation in the promoter completely disrupts the RNA polymerase binding site, are the genes in the trp operon transcribed, translated, both or neither?
None of the genes are transcribed or translated
If a mutation disrupts a critical codon in the trpE gene, is it transcribed, is it translated, and is tryptophan produced? If a mutation disrupts a critical codon in the trpR gene, are the genes in the trp operon expressed?
- all the genes will be transcribed and translated, although trpE will not be transcribed and translated correctly, therefore tryptophan is NOT produced
- The genes will always be expressed
If a mutation critically disrupts the operator sequence of the trp promoter, but RNA polymerase can still bind, are the genes in the trp operon expressed in the presence and absence of tryptophan?
Yes
If a base pair insertion occurs in the promoter, but this does not affect the binding of RNA polymerase or the repressor, is there a frameshift in the coding sequence?
No
If a base pair insertion occurs in the trpE coding sequence, are the genes transcribed, or translated? Is there a frameshift only in trpE, or a frameshift mutation in all proteins?
- Genes will be transcribed and translated (although trpE won’t be correct)
- Frameshift only occurs in trpE (remember Shine-Dalgarno sequence)
What is the function of the genes in the lac operon? (in general) What is the operator for?
- To break down lactose to use as an energy source
- Used to stop lac genes being expressed when there is no lactose (and when allolactose does not bind to it)
What protein is produced by the lacI gene, and what does it do?
Active lac repressor which binds to the operator to block gene expression in the lac operon (in the absence of lactose)
What molecule binds to the lac repressor? Where does this molecule come from (why is it around)? Does the binding of this molecule make the protein more active or less active?
- Allolactose
- The breakdown of lactose in the cell
- Makes it more active
Is the lac operon expressed in the presence or absence of lactose?
Presence of lactose
Which of the trp operon and lac operon is repressible and which is inducible? Why are they called this? Do each of the operons show negative regulation or positive regulation?
- trp operon: repressible - is activated when a molecule binds to it
- lac operon: inducible - is deactivated when a molecule binds to it
- Both show negative regulation (they are both repressors)
What does it mean to say that a gene is regulated with combinatorial control?
It means that a variety of regulatory sequences affect whether or not the gene is expressed and how much the gene expresses
What is cAMP? What is CAP? Where does CAP bind? What does it achieve when it binds?
- cAMP: cyclic AMP (from ATP)
- CAP: catabolite gene activator protein, binds to a CAP-binding site that is located in the promoter before the RNA polymerase binding site
- When CAP binds to the promoter, it activates the genes and greatly increases the likelihood of RNA polymerase binding and genes being expressed
What does binding of the lacI repressor do to the DNA of the lac promoter region?
It twists it into a loop, stopping the RNA polymerase so that it won’t transcribe
What are the characteristics of the DNA sequence of the lac operator that make it likely to be bound by a dimer? (note: for lacI, these dimers then bind together in a tetramer)
The two antiparallel sequences are palindromic (they are the same sequences running in opposite directions)
Do transcriptional regulators bind to DNA, RNA, or the protein?
DNA
What is the quaternary structure of most transcriptional regulatory proteins (specific transcription factors)?
- Many are dimers (Helix loop helix)
- Some have varying zinc finger domains
What is a common pattern for the sequences that transcriptional regulators bind? How does the pattern of binding sequences and the quaternary structure of the transcriptional regulators relate?
- ???????????
- ASK VERKADE OR SOMEONE ELSE
- THE FUCK IS WITH THE WORDING OF ALL THESE QUESTIONS!?
When a transcriptional regulatory protein binds to DNA, does it bind to the phosphates, the sugars or the bases? Why is this important?
The bases. Because it recognises the different shapes and charges in the major groove of the different bases
Why is the major groove a better binding target for proteins than the minor groove? Think about how the protein ‘reads’ the sequence information from the DNA
Because all of the shapes and charges of each base bindings are different in the major groove
What sort of chemical bonds hold the transcriptional regulatory protein to the DNA?
Hydrogen bonds
What is an enhancer? An activator? What is a mediator? Is mediator one protein? What is the function of mediator?
- Enhancer (AKA enhancer elements): DNA sequence that activators bind to
- Silencers: Where repressors bind (not as common as enhancers)
- Activators: Proteins that encourage DNA transcription
- Mediator: Large protein complex that mediates between the regulatory transcription factors and general transcription factors (TBP and TFIIB) due to large distances between the sequences
Give two examples of DNA binding domains found in eukaryotic transcriptional regulators.
- Zinc finger domains
- Helix loop helix domains
What is a zinc finger and why does it have zinc in its name?
a zinc finger is a eukaryotic DNA binding domain, it has zinc in its name because a zinc ion is coordinated in the protein
The initial pre-mRNA transcript is edited to make the mature mRNA. What does this mean? What does the editing? Is this process regulated?
Introns and some exons are spliced out by the spliceosome in the CTD. Like all processes, this is also regulated.
For a single gene, is this editing of the pre-mRNA the same in all cells, or different?
Different in different cells
Different proteins are produced from the α-tropomyosin gene in different types of muscle. How is this achieved?
Because different exons are kept, as in, along with the introns, some exons are spliced out as well to create different proteins
What does a splicing repressor do? What does a splicing enhancer do?
- Splicing repressor: prevents the recognition of a 3’-splice junction, so the spliceosome does not recognise the gene and recognises the next 3’ gene and splices the exon in the middle
- Splicing enhancer: enhances recognition of poorly recognised junctions
What is a miRNA? What is the protein complex that binds it? Together, do they bind DNA, RNA or protein? What part of the complex (miRNA or proteins) allows it to recognise and bind to this molecule? What happens to the molecule they are binding to?
- miRNA: micro RNA
- RISC: binds to miRNA
- miRNA + RISC: binds to RNA
- miRNA recognises the complementary structure in the RNA and binds to it with RISC
- The mRNA is either cleaved or the translation is repressed. Either way, translation does not occur.
Write a short section of a gene sequence. Write both strands, label the ends and label the strands. Identify where the promoter sequences would be. Figure out the sequence of a miRNA that could regulate the expression of this gene.
Give three examples of posttranscriptional modification, and what each achieves.
- Phosphorylation: acts as a reversible switch (activates and deactivates); induces protein-protein interaction
- Glycosylation: folding stability and extracellular functions
- Palmitoylation: Allows protein clustering at the membrane for synapse function (neurons)
- Formation of disulphide bridges: Folding and stability
- Ubiquitination: Addition of ubiquitin signals protein for things like degradation
- SUMOylation: addition of SUMO protein - changes localisation or binding partners
How many chromosomes do you have?
46 chromosomes (22 pairs of autosomes and 2 sex chromosomes)
How many different types of chromosomes does a human male have? A human female?
- MALE: 22 autosome pairs + 1 X and 1 Y chromosome = 24 different chromosomes
- FEMALE: 22 autosome + 1 pair of X chromosomes = 23 different chromosomes
How many chromosomes did you inherit from your father? From your mother?
23 chromosomes from each parent (22 autosomes and a sex chromosome)
What are the major landmarks on a chromosome (there are two) and what do they do?
- Centromere: Controls movement of chromosomes during cell replication
- Telomere: protects from being shortened for every round of replication
How does the packing of chromosomes change at different stages of the cell cycle?
Chromosomes are usually relaxed/unwound during most of the cell cycle (interphase)
It only becomes more condensed during G2 and is at its most condensed during mitosis
Is DNA in the cell generally underwound or overwound? What does this mean?
Generally underwound, therefore the DNA is generally under tension (which means it is more likely to supercoil or the strands are more likely to separate)
What is a DNA supercoil?
DNA coiling to relieve tension in underwound DNA
How does supercoiling affect the running of a circular DNA on an agarose gel?
The more supercoiled, the faster it runs through the agarose gel
What does topoisomerase do to the structure (base pairs) of DNA? To the shape? To the size?
What is the general mechanism (no detail required) by which Type II topoisomerase acts?
What is a nucleosome? What is chromatin?
Where are the 5 types of histones found in chromatin? What is the (overall) structure of a nucleosome? What is important about the histone N-terminal tails?
Name three types of histone modification that can be used to regulate chromatin packing.
What is meant by the ‘histone code’?
What is a 30nm fibre?
What is the difference in chromatin packing between a part of the DNA that is not being accessed by proteins, and a part of the DNA that is being actively used (eg. actively transcribed)? Does acetylation of histones open the chromatin structure or tighten it?
Which DNA base is chemically modified in the regulation of gene expression? Is gene expression increased or decreased because of it?
