Med 1002 (II) Flashcards
What is a distinguishing feature of a chromosome always?
- Has telomeres
- Has a centromere
What is the role of microtubules in chromosome replication?
Microtubules pull apart the sister chromatids so each cell gets a chromosome
What is the role of kinetochores in chromosome replication?
Two of them are attached on either side of the centromere. Micro tubules attach to the kinetochore
Describe the polarity of microtubules in regards to chromosome replication
Microtubules attach at two ‘poles’ with the positive end being at the kinetochores and the negative being at the centrosome
Describe the function, structure of a centrosome and any other names it may have
- Also known as spindle poles and microtubule organising centre
- One per cell but when cell is going to split, we have two of them so chromosome can get pulled apart
- It’s made of two centrioles which are right angles to each other
What are the two kinds of microtubules?
- Kinetochore microtubules
- Attach microtubules to centrosomes
- Astral microtubules
- Go from centrosome to cell membrane
Explain what disjunction is in regards to chromosome replication
Very important to get chromosomes properly
separated into 2 new cells = segregation.
Incorrect segregation = nondisjunction
List all the phases in the cycle
Interphase (growth and normal phase):
- G0
- G1
- S
- G2
Mitotic (division phase):
- Prophase
- Prometaphase
- Metaphase
- Anaphase
- Telophase
Detail what occurs in G0
- when a cell stops replicating.
- Not all cells undergo G0 and some cells can come out of G0 back to G1
Detail what occurs in G1
- Cell grows
- Normal metabolic reactions occur
- Cell prepares for S phase
Detail what occurs in S phase
- DNA replication occurs (amount of DNA doubles)
- Centrosome replication is completed (we now have 2)
- Nucleus becomes enlarged
Detail what occurs in G2
- Further growth of cell & protein synthesis
- Centrosome maturation
- Cell begins to divide
Detail what occurs in prophase
Chromosomes condense. Each chromosome has two chromatids and mitotic spindle (tubules) form
Detail what occurs in prometaphase
- Nuclear membrane disintegrates
- Spindle microtubules attach to chromatids
Detail what occurs in metaphase
Chromosomes line up on the metaphase plate (looks like they’re in the centre of the cell)
Detail what occurs in anaphase
Sister chromatids separate and move towards opposite poles or sides of the cell
Detail what occurs in telophase
- Chromosomes arrive at spindle poles
- Nuclear membrane reforms
- Chromosomes relax
How does the number of chromosomes in a cell change as mitosis occurs?
Until anaphase, there are always 4 chromosomes per cell
How does the number of DNA molecules in a cell change as mitosis occurs?
Always 8 DNA molecules except in S phase (where it goes from to 8), G1 and telophase
What are the differences between meiosis and mitosis?
Meiosis occurs in sex cells and mitosis in somatic cells.
In meiosis, the daughter cells have half the amount of chromosomes as the parent cell
What are the differences between meiosis and mitosis?
Meiosis occurs in sex cells and mitosis in somatic cells.
In meiosis, the daughter cells have half the amount of chromosomes as the parent cell
Detail what occurs during prophase I
Chromosomes begin to condense, spindle forms, homologous pairs of chromosomes pair up and in late prophase, crossing over occurs between chromosomes
Detail what occurs during metaphase I
Homologous pairs of chromosomes line up on metaphase plate
What occurs during anaphase I
Homologous chromosomes separate and move towards separate poles
What occurs during telophase I
Chromosomes arrive at spindle poles and cytoplasm divides
Detail what occurs in mitosis II
(prophase, metaphase, anaphase and telophase II only)
- Chromosomes recondense
- Individual chromosomes line up on metaphase plate
- Chromosomes separate and then cytokinesis happens
What is crossing over (cell cycle)?
Exchange of genetic material between chromosomes in meiosis I.
Crossing over occurs at chiasma (sites of crossing over)
What is the role of cohesin and shugoshin?
In mitosis, cohesin keeps sister chromatids together. Breaks down during anaphase.
In meiosis, cohesin holds chromosome arms together at chiasmata in meiosis 1. Shugoshin protects cohesin at sister chromatids (at centromere) and shugoshin and cohesin degrade during anaphase 2 (meiosis II)
What regions of chromosomes replicate first and which regions replicate later?
Less condensed regions (euchromatin) replicate before more condensed regions (heterochromatin)
What governs chromosome replication?
- Control of initiation. This is related to the sequences of DNA so which origins are used and when they are used is dependent on the structure and activity of chromatin.
What is a replication bubble?
Replication forks occur in pairs, forming a bubble
What is an origin on a chromosome?
A precise location on a chromosome where assembly of replication protein occurs. They are A-T rich. There are multiple origins on a chromosome
What is ORC?
Origin replication complex, which binds to origin and separates DNA strands by recruiting replication proteins
When is DNA replicated in the cell cycle?
S phase
What is chromosome licensing?
At different origins, DNA and the origin is licensed ONLY once during a cell cycle to initiate replication and by initiating only once, ensures replication of DNA only occurs in S phase
What occurs at the telomeres at the end of chromosome replication?
The telomeres of newly synthesised DNA would shorten because primer at the ends leaves gaps which ligase cannot join together.
How is telomere shortening prevented?
A specialised enzyme, telomerase, with a catalytic subunit = Telomerase Reverse Transcriptase (TERT) attaches to the parent strand and then extends it which allows primer to be laid. This allows more replication to occur because you need to attach nucleotides at 3’ OH group. The overhang is then chopped of by FEN 1(not sure if it’s FEN1)
What is the role of CAF-1?
Brings histone proteins to replication forks to assemble nucleosomes and then chromosomes
Describe the general properties and structure of RNA
Comprised of sugar, phosphate group and nitrogenous base. Sugar is ribose sugar compared to DNA’s deoxy ribose sugars. Instead of thymine, we have uracil complementary to adenine. RNA is also be single stranded
What is the first degree of structure of RNA comprised of?
The sequence of ribonucleotides
What is the second degree of RNA comprised of?
Complex structures arising from intra-strand base pairing via H-bonds
Explain the effects of RNA’s secondary structure
Due to complex secondary structure, RNA can allow for various functions.
It can base pair conventionally (A-U) but also unusually such as U-U (however they’re weaker)
As RNA can fold, it can have enzyme activity or structural roles
Compare and contrast U=G and U=A hydrogen bonding in RNA
U=G because they’re not complementary bases don’t have 3 hydrogen bonds so it’s weaker than U=A because base stacking doesn’t properly occur either
Where is all RNA synthesised/transcribed
Within the nucleus
Where is rRNA synthesized and explain how it is synthesized?
Synthesized in nucleolus from two genes.
It has two parts:
- Three RNA different sized molecules, 18S, 5.8S, 28S
- 5S unit which is from a different gene from previous subunit
These two subunits combine to form proper RNA
What are the two classes of RNA?
Coding and non-coding
List the 3 kinds of RNA essential to protein synthesis
mRNA (messenger RNA)
rRNA (ribosomal RNA)
tRNA (transfer RNA)
Explain the role of mRNA
A messenger RNA that carries the instructions of amino acid sequences. mRNA goes to the ribosome where protein synthesis occurs
Explain the role of tRNA
They form the structural and functional part of the ribosome, so they are the site of protein synthesis. They translate information from mRNA into AA sequence of protein.
- They also transfer amino acids to the ribosome and bind mRNA by matching their anticodon to mRNA codon
Explain what is ribozyme
Ribosomes essentially catalyse peptide bond formation so they need a catalytic component which is ribozyme
What is the role of the anticodon?
Binds mRNA via complementary base pairings (RNA is anti parallel)
What is the purpose of having both DNA and RNA?
DNA is more stable and less reactive than RNA as the deoxyribose is more stable. Also errors in DNA are less due to proofreading by polymerases.
What is the structure of a protein encoding gene/transcription unit?
Regulatory region: Contain sequences that are recognised and bound by proteins that transcribe DNA into RNA. One before and after transcribed region
Transcribed region: Contain information for the nucleotide sequence of gene product.
- Region close to transcriptional start site is called promotor
- Region far away from transcriptional start site is called enhancer
What is upstream vs downstream region of DNA?
Upstream is closer to 5’ end, downstream is closer to 3’ end
Describe which of the DNA strands are used for DNA transcribing?
Either the code/sense strand or the template/antisense strand but not both for transcribing a single gene.
What are some conventions when it comes to talking about RNA and genes?
Gene is always considered 5’ to 3’
RNA is antiparallel to DNA
Start of transcription is called +1 which is the start of the RNA
Upstream (towards 5’) promoter region is known as -1
RNA sequence is the same as coding strand (U instead of T)
The transcribed RNA is called the PRIMARY TRANSCRIPT
What are the basic steps in RNA transcribing?
- Initiation: Deciding when and where to start
- Elongation: Polymerizing ribonucleotides into a RNA molecule
- Termination: Deciding where to stop
What enzyme is responsible for Pre-mRNA synthesis?
RNA polymerase II
Describe the structure of RNA polymerases, particularly RNA polymerase II
RNA pol I, II and III have 10 common subunit and 7 unique subunits.
Formed by large complex proteins
RNA polymerase II has a tail called CTD (Carboxyl terminal domain) which can be phosphorylated or dephosphorylated which impacts shape
Explain how RNA synthesis is initiated
RNA polymerase binds to promoter sequence in DNA. Then the pol separates the DNA near the transcription start site (only one DNA strand needs to be used), which forms a transcription bubble. Then the pol catalyses phosphodiester linking between TWO initial ribonucleotides
Explain how RNA elongation occurs
RNA polymerase adds nucleotides 5’ to 3’ direction by separating the two strands of DNA along the way
Explain how RNA termination occurs
At transcription end site, polymerases releases and disassociates from DNA
What are the two kinds of promoter regions that exist?
Regulatory promoter and core promotor
What is the role of a core promotor?
A core promotor has elements which help to attach polymerase and provide a site to which RNA transcription can begin.
What are the 4 kinds of of core promoters that can occur in RNA polymerase II transcribed genes?
TFIIB recognition element (-35 or 30 ish nucleotides upstream of initiation site)
TATA box (-25)
Initiator element (+1)
Downstream core promoter element (+30)
Describe the TATA box
As the name suggests, repeating sequence of A and T nucleotides. Used in genes that need to rapidly transcribe.
Describe the steps in RNA transcription initiation
- TBP, a TATA binding protein which is part of TFIID, binds to TATA box and bends the DNA
- TFIIB binds and makes contact with both TBP and the DNA
- A complex of TFIIF and RNA Pol II binds so that Pol II is positioned over start site
- TFIIE binds and forms docking site for TFIIH
- TFIIH binds as 2 subunits that have helicase activity
- TFIIH unwinds DNA which uses ATP, at start site
What are all the TFIIs about?
They are general transcription factors for RNA pol II
What is the role of the regulatory promoter?
It provides a spot for some proteins to bind to and helps build transcription machinery
What are the processes involved with mRNA?
RNA capping at 5’ end
Polyadenylation (adding A bases) at 3’ end
RNA splicing
What does it mean when we say mRNA processing is co-transcriptional?
All the processes involved in mRNA processing occur at the same time rather than one after another
What do we mean by untranslated regions in mRNA and what is their purpose?
These regions are regions which is transcribed into the mRNA but is not translated into proteins. They control translation initiation
What is RNA capping (5’)?
In mRNA, 7-methylguanosine is added to primary transcript (main mRNA) via 5’ to 5’ triphosphate linkage (not a phosphodiester linkage)
What is the purpose of RNA capping? (5’)
Protects against nucleases which may break down RNA in 5’ to 3’ direction
Facilitates transport of mRNA from nucleus to cytoplasm
Facilitates RNA splicing
Has role in translation initiation
What is a role of the CTD and where is CTD found?
CTD or carboxyl terminal domain is the tail of RNA polymerase II. CTD-P (phosphate attached), binds enzymes and factors for capping and splicing of mRNA
What is the role of polyA polymerase?
In mRNA adenylation, polyA polymerase adds a bunch of A’s at 3’ end forming a poly(A) tail
What is the role of the consensus sequence in mRNA?
It signals that 11-30 nucleotides further downstream, cleavage of the RNA needs to occur so polyadenylation can begin.
What kind of sequence is cleaved and degraded from mRNA during polyadenylation?
GU repeat units or U-rich sequence
Why is polyadenylation important?
Allows for mRNA export and better stability while playing role in translation
What are introns and exons?
Areas within the transcribed region of the gene. Exons encode for proteins while introns are spliced out as they are non coding
What are the three sequences/sites required for RNA splicing?
At the boundary of introns and exons, there are these sequences of bases which signal to spliceosomes where to catalyse splicing reactions. At the 5’ end of intron we have GU (splice donor site) , at 3’ end of intron we have AG (splice acceptor site)
We also have a branch point which is nucleotide with A base
Describe the process of mRNA splicing
- mRNA is cut at 5’, GU, donor site of intron
- This end now of the intron now binds to the branch point
- mRNA is cut at 5’, AG, acceptor site of intron
- Intron now released as a LARIAT
- As intron released, exons now spliced together
- The intron degrades and exons exported to cytoplasm to be translated
What is a spliceosome and what is its function?
It’s an snRNP (small nuclear ribonucleoprotein) which itself is a complex of snRNA and proteins.
snRNA catalyses splicing and is a ribozyme
What is a RNA lariat and how is it formed?
Formed when intron is spliced out during transcription. It’s an intermediate RNA.
RNA lariat formed by joining 5’, GU, end of intron and A branch point. 2’ OH group of branch point and 5’ Phosphate of intron form a covalent bond VIA NUCLEOPHILIC ATTACK
How are two exons joined together after splicing out introns?
Phosphodiester bond
What is alternative splicing?
From the exact same gene, the exons are joined together in different combinations so mRNA and thus protein is different. Usually tissue specific
How many introns do you have for exons?
Look at your hand. For 5 fingers, we have 4 spaces so similarly we have one less intron for every exon
What are the ways alternative splicing can occur?
Either in a RNA lariat we can have an exon just be degraded with it.
Or an exon can be cleaved during adenylation to be bigger or smaller so the last exon in mRNA can be bigger and smaller depending on how it’s spliced and the cleavage site.
Where does all the transcription and processing of mRNA occur
Nucleus
What is translation?
Forming proteins from mRNA messages by linking amino acids with peptide bonds
What is a codon?
3 nucleotides of a sequence which is read during translation.
In translation, sequence is read as codons or groups of 3 nucleotides.
What is significant about methionine?
Methionine or Met, is always the first amino acid made during translation. It is coded by the codon AUG BUT IT CAN BE REMOVED LATER, NOT EVERY AMINO ACID HAS MET AS FIRST ACTUALLY, JUST FIRST TRANSLATED
What are stop codons?
3 codons which DONT encode for an amino acid, just terminate translation
What do we mean when we say genetic code is degenerate?
20 amino acids and 64 (4^3) codon combinations so multiple codons can lead to different amino acids but one codon always is linked to one amino acid.
Note: Only Met and Trp have only one unique codon
What do we mean when we say genetic code is non overlapping?
Basically, one codon encodes for one amino acid and then it moves to another codon without taking in a previous codon’s nucleotides
What are reading frames in genetic code?
Essentially where the reading starts from, maybe in one codon instead of starting reading at first nucleotide, it starts from position 2 or 3.
Note: Stop codons are often found in non-coding frames to ensure correct reading frame is being used
Which direction are proteins made in during translation?
Amino to carboxy
How is tRNA attached to it’s base?
Each tRNA molecule has different anticodon loop. It has different 3-D structure too. Enzymes known as aminoacyl - tRNA synthetases bind to specific amino acids and tRNA molecules (they know this because of 3-D shape). The base is attached to the 3’ end and is ALWAYS connected to an A base
What is the structure of a ribosome?
Large and small subunit, large one sits on top of small one. Within them, they have 3 sites:
Exit (E)
Peptidyl (P)
Aminoacyl (A)
These sits go through both large and small subunits
The small subunits is the mRNA binding site
What is the wobble hypothesis?
You have multiple codons coding for different amino acids. Third position in anticodon can Watson-Crick base pair weirdly (G-U instead of G-C). It’s usually this third position as the first two nucleotides are common when coding for different. Note: U/G will tend to unconventionally base pair, A/C won’t
How is translation initiation done?
We need to find AUG codon. Requires eukaryotic initiation factors. We are looking for Met-tRNA to bind to AUG. The small subunit of ribosome, the P site has met-tRNA and goes along mRNA to find AUG and when found, starts translating.
Initiation is affected by poly (A) tail at 3’ end and cap at 5’.
After finding AUG, initiation factors disassociate, large ribosomal unit binds, and elongation occurs.
Describe how translation and protein elongation occurs after initiation
Three sites, E, P and A. tRNA comes to A site and sees if anticodon and codons match, if not then ejected but if they match, binds to A-site. A peptide bond is formed with the amino acid currently in the P site. Then, large subunit physically moves, causing P site tRNA to move to E and A to P. The E site tRNA is now released, and small subunit moves along 3 NTS along mRNA and process repeats.
What are polyribosomes/polysomes?
Usually multiple rounds of protein translation occur. Polysomes are multiple ribosomes which TRANSLATE THE SAME mRNA so they produce a lot of same protein.
What is the roles of probes with regards to DNA?
Probes bind to certain DNA molecules, either homologous molecules (directly complementary) or heterologous (aren’t fully complementary)
Describe how southern, northern and western blotting occurs and the differences between them.
Protein, DNA or RNA is broken down into fragments or stays whole. First, gel electrophoresis is used to separate different fragments as smaller molecules move further throughout the gel. Then either using pressure or charge, results are put onto a membrane and a probe is used with something that can visibly show the results. Probe shows us if target molecule is present and detection molecule attached allows us to see it.
Southern blotting: DNA
Northern blotting: RNA
Western blotting: Protein
DNA and RNA use probes while proteins need specific antibodies
What would happen if a translation error occurs?
The sequence of a protein is affected
What would happen if a transcription error occurs?
mRNA and subsequent protein is affected
What would happen if DNA replication error occurs?
DNA, RNA and protein affected. Also, this is passed down to daughter cells
What is polymorphism?
Natural variations in DNA that have no adverse effects on the individual and occur with fairly high frequency in general population
What are alleles?
Alternate forms of a gene (nts may be different)
What is a phenotype?
The physical trait something has due to a gene
Compare and contrast wild type genes vs variant genes
Wild type is standard and found in nature. Variants are altered genes or characteristics
What is a substitution variant of DNA?
A base is replaced by another base
What is a deletion variant of DNA?
One or more base pairs is lost
What is a insertion variant of DNA?
One or more base pairs is added
What is inversion variant of DNA?
180 degrees rotation of piece of DNA
What is chromosomal rearrangement variant of DNA?
Entire chromosome is rearranged
What is translocation of DNA?
Kind of variant in DNA where segment of chromosome has changed place
What are the two kinds of base substitution?
Transition: Purine substituted by purine (same with pyrimidine)
Transversion: Pyrimidine substituted by purine and vice versa
What are point variants and what kinds of point variants occur?
DNA mutations where only one nucleotide is changed.
Synonomous: (when alteration leads to same amino acid because of reduduncy)
Missense: Two types
Conservative: Similar amino acid based on chemical properties translated
Non-conservative: Completely different amino acid translated
Nonsense: (when alteration to stop codon and termination occurs)
Frameshift: When insertion or deletion (indel) leads to frame reading to not be correct.
How can incorrect splicing of RNA lead to issues?
Exons can be:
Deleted (exon skipping)
Shortened
A new exon which isn’t supposed to be there can be added
Leads to incorrect expression of gene
What is the difference between forward and reverse mutation?
Wild type -> variant/mutant = forward
Variant/mutant -> Wild type = reverse
What are the two kinds of reverse mutation?
Exact reversion: When variant goes to the proper wild type sequence
Equivalent reversion: Different DNA sequence but the proper amino acid still gets there because of redunduncy
What are the effects of point variants phenotypically?
They can change pre much anything but phenotypically they don’t always change stuff
In variant nomenclature, what nucleotide is assigned position #1?
From the ATG codon, the A has +1. Anything before has negative, anything after has positive
In variant nomenclature, how do we describe a substitution in DNA?
c. 512G>A
c. means coding strand
Number is nucleotide number
> means the one before this symbol substituted the one after it
In variant nomenclature, how do we describe indel of DNA?
Deletion: c.197_198delAG (AG deleted)
Insertion: c.2552insT (T inserted)
In variant nomenclature, how do we describe changes in amino acids?
p.Arg197Gly
p. indicates protein, number is position
What are the two kinds of origins of variants in DNA/protein?
Spontaneous variation: No mutagen and just occurs randomly
Induced variation: Prescence of mutagen, greater dose = more change
What is replication slippage?
A spontaneous variation where in sequences with lots of repeat units (AAAA, UAUAUA etc.) causes little bends in coding and template strand. These bent bases dont Watson-Crick base pair with anything so leads to indel on new strand.
What is trinucleotide repeat disorder and give an example?
When in a daughter strand, you get a repeat of 3 acid base pairs (3 nucleotide pairing is repeated). Huntington’s (CAG trinucleotide is repeated in the coding region of gene for Huntingtin) and Fragile X syndrome are examples of this
How do we differentiate between genes and proteins when both have same name?
CFTR gene and protein:
Gene CFTR has capital letter and italics
Protein CFTR has capital letters and no italics
