Gauvrit section post midterm Flashcards
should you go back and do the first lecture in this section?
yes
What is DNA supercoiling?
DNA that is more compact than its relaxed counterpart is supercoiled
- Underwound DNA is negatively supercoiled
- Overwound DNA is positively supercoiled
- negative supercoiling is important to allow chromosomes to fit within nucleus
What are the two kinds of topoisomerases?
- Change the level of DNA supercoiling
- Type I - change the supercoiled state by creating a transient break in one strand of the duplex
- Type II - make a transient break in both strands of DNA duplex - can tie or untie knots - can interlink or separate circles
What is DNA denaturation?
Ability to sperate into components
- thermal denaturation
- more GC means higher melting point
What is nucleic acid hybridization?
complementary strands of nucleic acids from different sources can form hybrid molecules - important for sequencing and cloning
What are the three broad classes of DNA sequences?
- classes by how often their nucleotide sequence is repeated
- highly repeated fraction
- moderately repeated fraction
-nonrepeated fraction
What are three common highly repeated DNA sequences? How much of our total DNA are they?
1-10% of total DNA
- Satellite DNA - short sequences that evolve rapidly
- Minisatellite DNA - unstable and tend to be variable in the population - DNA fingerprinting
- Microsatellite DNA - shortest sequences and typically found in small clusters - found in genetic disorders
What is FISH?
Fluorescence in situ hybridization
- used to determine location of a DNA sequence within a genome
- can be used to visualize repetitive sequences like satellite DNA found in the centromeric regions of the chromosome - or to determine the position of single copy genes
What are moderately repeated DNA sequences? How much of our total DNA do they make up?
Varies greatly - between 20 and 80% of DNA
- some sequences code for abundant gene products like RNA but most lack a coding function
- noncoding elements are scattered throughout the genome and can be grouped into SINES or LINES
What are SINES and LINES?
Short interspersed elements and long interspersed elements
What are nonrepeated DNA sequences?
single copy DNA sequences invlude genes that exhibit mendelian patterns of inheritance and localize to a particular site on a particular chromosome
- includes coding sequences for virtually all proteins other than histones - less than 1.5% of genome
- even though sequences are only in one copy - genes that code for polypeptides are usually members of a family of related genes like globins actins etc
What is polyploidization?
Whole genome duplication - offspring have four chromosome homologues rather than 2
- often done to produce bigger fruits
-
What is the globin family and how did these genes evolve?
Hemoglobin, myoglobin, plant leghemoglobin
- ancestral forms have given rise to modern forms by duplication fusion and divergence
- some sequences called pseudogenes resemble globin genes but are nonfunctional
What do “jumping genes” refer to?
genetic rearrangement of genetic elements called transposition - via transposable elements
- only certain sequences can act on transposons
- requires enzyme transposase
- retrotransposons use RNA which produces complementary DNA vs reverse transcriptase
How are jumping genes important in adaptive genome evolution?
- transposable elements can carry parts of the host genome with them as they move from one site to another
- transposable elements themselves appear to have given rise to genes
How many genes does the human genome have?
20 000
What is alternative splicing?
when a single gene can encode a number of related proteins
is a significant proportion of functional DNA conserved?
no they are constantly evolving
The majority of the genome lies between ____ and its termed ____
lies between protein coding genes and is termed intergenic
- much of the 21000 protein coding regions consist of non coding portions (intronic DNA)
What are three genes that are relevant in humans and chimps
FOXP2 - similar in humans and chimps -speech gene
HAR1 - similar between humans and chimps - function unknown
AMY1 - amylase enzyme - frequency is very different
What is the exome?
portion of the genome that codes for proteins
What are the three parts of a chromosome?
- chromatin - fibers composed of DNA and proteins
- histones - highly conserved proteins
- nucleosomes - the subunits that DNA and histones are organized into
Explain the structure of a nucleosome complex;
Histone H1 serves as a linker
- DNAis wrapped around the core complex
- the core complex is 2 H2A, H2B, H3, H4 - forming an octomer
-
What groove faces the histone core?
minor groove
What is important about the histone tail?
- each core histone has a flexible N term tail that projects beyond the DNA helix
- histone tails can be modified
What are chromatin loops?
Higher level chromatin structure
- takes 30nm chromatin fibers and gathers them into large 80-100nm supercoiled loops
- DNA loops are tethered at their bases to proteins
-
What does cohesin do?
-holds replicated DNA molecules together during mitosis
- maintains chromatin loops
What is euchromatin?
Chromatin that returns to a dispersed state after mitosis in interphase
What is constitutive and facultative heterochromatin?
Constitutive - remains condensed all the time - found mostly around centromeres and telomeres and consists of highly repeated sequences
Facultative - inactivated during certain phases of the organisms life - one of the X chromosomes as a Barr Body through X inactivation
What is a Barr body?
condensed inactive X chromosome in females - to preserve ratios
What is epigenetics?
study of how your behaviours and environment can cause changes that affect the way your genes work - not changes in genes itself but how body reads the genes and expresses them
What is the histone code hypothesis?
The histone code hypothesis states that the activity of a chromatin region depends on the degree of chemical
modification of histone tails
What are the two things histone tail modifications can do?
Can serve as docking sites to recruit nonhistone proteins
Can alter the way histones of neighboring nucleosomes interact
Where is the majority of modified amino acids found in histones?
N terminus of H3 and H4
What inactivates H3 histones?
methylation - stability
removal of acetyl groups from H3 and H4 convert to heterochromatin
- Inactive heterochromatic X chromosome has deacylated histones
What is a telomere?
repeated sequence on DNA tips that with a group of specialized proteins form a cap called a telomere
Why are telomeres used?
to get around the end replication problem - RNA primer causes the removal of some DNA at the 5’ end
- telomerase is a reverse transcriptase that makes DNA from RNA that the enzyme itself has - once end has lengthenes the DNA polymerase can return complementary strand to previous length
What is the significance of telomeres?
- required for complete chromosome replication
- form caps that protect chromosomes from nucleases
- prevent ends of chromosomes from fusing with one another
What is the centromere?
indented site on chromosome that is repeated heterochromatin
- has centromeric DNA where microtubules attach during mitosis
What is RNA folding driven by?
formation of regions having complementary base pairs
What are transcription enzymes also known as?
RNA polymerases
What direction does RNA polymerase move in? How fast does it assemble the complementary strand?
Moves in the ????? (3’ to 5’) direction - roughly 20-50 nucleotides a second.
What is different about bacterial transcription and translation?
They occur at the same time - there is no physical separation of DNA/RNA/ribosomes
How are genes organized in bacteria?
organized in clusters that are transcribed together as a single mRNA molecule encoding for multiple proteins - an operon
What do transcription factors do?
assist in beginning transcription and making sure rna polymerase is working
How many RNA polymerases do bacteria have?
one - 5 subunits
Where does transcription and RNA processing occur in eukaryotes?
in the nucleus
What is a holoenzyme?
complete enzyme with core enzyme, sigma unit and promoter region
Where does translation occur in eukaryotic cells?
cytoplasm
what binds the promoter
sigma subunit (?)
What are the three types of RNA polymerase in eukaryotes and what do they transcribe?
Most rRNAs are transcribed by RNA polymerase I
-mRNAs are transcribed by RNA polymerase II
- tRNA are transcribed by RNA polymerase III
What is the most common type of RNA?
mRNA
What direction does RNA polymerase incorporate nucleotides?
5-3
In transcription a 9nt what is formed?
DNA RNA transient hybrid
Transcription generates ___ DNA ahead and ___ behind
overwound (positively supercoiled) and underwound (negatively supercoiled)
What are transcription factors?
proteins that bind DNA, each recognizes different DNA sequences - controlling the rate of transcription.
RNA polymerase II is assisted by _________ to form the ________
general transcription factors (GTFs) to form the preinitiation complex (PIC)
What does BRE mean?
B recognition element
What does DPE mean?
downstream promoter element
What is on either end of an mRNA molecule?
3’ poly A tail, 5’ cap
eukaryotic genes contain ___ which are missing from mature mRNAs
intervening sequences
The presence of genes with intervening sequences are called ??
split genes
What are exons and introns?
exons are coding sequences that are present in the final mRNA, introns are removed during mRNA processing
What are RNA transcripts associated with after they are synthesized?
ribonucleoproteins
Pre mRNA transcripts are processed ___
cotranscriptionally
What is the point of introns?
thought to increase number of proteins made per gene - able to remove specific sections to encode for different proteins.
Transcription factors bind to (2)
- bind at core promoter sites in association with RNA pool
- bind to various regulatory sites of particular genes (can act as transcription activators or inhibitors)
What two domains do transcription factors contain?
- DNA binding domain
- activation domain
what kind of cells are capable of indefinite self renewal?
embryonic stem cells
More than 80% of the RNA in most cells consists of ____ RNA
ribosomal (rRNA)
What is the size of the bacterial ribosome and subunits
70s, 50s large, 30s small
What is the size of the eukaryotic ribosome and subunits?
80s,, 60s large and 40s small
What are ribosomal subunits made of?
rRNA and proteins
How is mammalian ribosomal RNA processed (complicated)
- pre-rRNA generation
- the 120 nt 5S rRNA is synthesized by RNAPolIII
-the 45S precursor is synthesized by RNAPolI - cleaved to 28S, 18S and 5.8S rRNA
-5 “cut” locations - first cut at site 1 and 5, then 2 or 3
What is the site of ribosome biogenesis?
Nucleoli
How is the 45S pre rRNA processed?
posttranscriptionally
What carries out the processing of pre rRNA?
snoRNPs (small nucleolar ribonucleoprotein particles) that begin to associate with the primary transcripts before transcription is completed.
What is present in all snoRNAs that guide ribose methylation?
BoxD, 5-CUGA-3
How are tRNAs made? Which rna polymerase
- synthesized from genes found in small clusters scattered around the genome
- transcribed by RNA polymerase III and the primary transcript is trimmed on its 5’ and 3’ sides
What endonuclease is involved in tRNA processing?
ribonuclease P
What is the start codon?
AUG - codes for Met/M
What are the stop codons?
UAA, UGA and UAG
What are missense mutations?
an amino acid specifying codon is replaced with a codon for a different amino acid
What are nonsense mutations?
an amino acid specifying codon is replaced by a premature stop codon
What decodes the information in an mRNA?
tRNA
What sequence do all mature tRNAs have?
triplet CCA sequence at 3’ end
What is the wobble base pair?
pairing between 2 nucleotides that does not follow base pair rules
- U of the anticodon can pair with A or G of the mRNA
-nucleotide of the 5’ end of the tRNA anticodon is capable of pairing with more than one nucleotide at the 3’ end of the mRNA codon
is energy required to add amino acids to each tRNA?
yes af
What do the small and large subunit do in translation?
- small subunit decodes the genetic message
- large subunit catalyzes peptide bond formation
What are the three stages in translation?
initiation, elongation, termination
How is translation initiated in eukaryotes? (complicated)
- initiator tRNA-Met complex is loaded into small subunit
- requires at least 12 initiation factors
- once the 43S preinitiation complex is formed it is ready to find the 5’mRNA
- small subunit moves 5-3 along mRNA to find start codon
- initiation factor dissociates
What is the 43S preinitiation complex?
40S subunit and initiator complexes together (i think)
What are the three places on the ribosome where tRNAs can bind?
A - Aminoacyl site binds to the incoming aminoacyl site carrying the new AA
P - Peptidyl site holds the tRNA with the protein
E- exit site holds the tRNA without AA which is then released by the ribosome
What happens in the first step of elongation during translation?
Step 1 - aminoacyl tRNA selection
- binding of the second aminoacyl tRNA to the A site requires the GTPase and elongation factor EF-Tu (bacteria)
- once bound to the mRNA codon - GTP is hydrolyzed and the EF-Tu-GDP complex released leaving the new AA-tRNA situated at ribosomes A site
What happens in the second step of elongation in translation?
Step 2 - peptide bond formation
- occurs spontaneously
- catalyzed by large subunit
- tRNA in P site has no AA while tRNA in A site has 2 AA
-What happens in the third step of elongation?
Step 3 - translocation
- binding of elongation factor and hydrolysis of its GTP results in the translocation of the ribosome
- as result of this motion, ribosome moves 3 nt along the mRNA in 5-3 direction
What happens in the 4th step of elongation?
Step 4 - releasing the deacylated tRNA
- leaves the ribosome emptying the E site
For each cycle of elongation how many molecules of GTP are hydrolyzed?
at least 2
What is termination in translation
occurs at stop codons
- dissociates mRNA from ribosome and disassembles ribosome
What is NMD? Nonsense mediated decay
nonsense mediated decay - mRNA surveillance mechanism capable of detecting messages with premature termination codons
- protects cell from producing nonfunctional shortened proteins
What is a polyribosome?
complex of multiple ribosomes on mRNA , allowing simulataneous translation
10% of all prescribed drugs act at the level of _____
gene expression
How is the bacterial genome organized?
- circular and double stranded
- nearly all DNA encodes RNA or proteins
-genes are often transcribed as an operon
What is an operon?
Functional complex of genes containing information for enzymes of a metabolic pathway
What 5 things do all operons contain?
- structural genes - code for enzymes
- promoter - where RNA polymerase binds
- Operator - site next to promoter where regulatory protein can bind
- Repressor - which binds specific sequences to determine wether or not a gene is transcribed - RNA polymerase cant bind promoter if repressor is bound
- Regulatory gene - encodes the repressor protein
What is the Trp operon?
- repressible operon - repressor cannot bind operator unless it has corepressor factor (Trp)
- if theres no tryptophan then repressor isn’t active and will transcribe genes
- once concentrations increase - will bind and repress / block transcription
What is the Lac operon>
- inducible operon
- turned on in the presence of lactose
- lactose binds to the repressor - making it unable to bind to the operator
- repressor protein can bidn to the operator and prevent transcription in the absence of lactose
What are the four levels of regulation of gene expression?
- transcriptional control
- processing control
- translational control
- posttranslational control
What is differential transcription?
- mechanism by which eukaryotic cells determine which proteins are synthesized
- occurs in cells at different stages of embryonic development
- occurs in cells in different tissues - tissue specific
- occurs in cells that are exposed to different types of stimuli
What are DNA microarrays?
- immobilized fragments of DNA are hybridized with fluorescent cDNAs - can monitor the expression of thousands of genes simultaneously
- genes that are expressed show up as fluorescent spots on immobilized genes
What can DNA microarrays and RNA sequencing accomplish in healthcare?
- personalized medicine for cancer therapies or treatment plans
What is the role of transcription factors in regulating gene expression?
- bind at core promoter sites in association with RNA polymerase (general transcription factors)
- bind to various regulatory sites of particular genes ( can act as transcription activators or inhibitors)
A single gene can be controlled by ___ ____ ____
different regulatory proteins
A single DNA binding protiein may control the expression of many different ____
genes
What two domains do transcription factors contain?
- DNA binding domain
- Activation domain
What are response elements?
- short sequences of DNA within a gene promoter or enhancer region that are able to bind specific transcription factors and regulate transcription of genes
What is an example of transcriptional activation discussed in class?
- the glucocorticoid receptor
- nuclear receptor that includes a ligand binding domain and a DNA binding transcription factor
- binds to a glucocorticoid response element which is a palindrome sequence
What are four DNA sites involved in regulating transcription?
- TATA box regulates initiation of transcription
- Core promoter from the TATA box to the start is where the initiation complex assembles
- the CAAT and GC Box are upstream and are required for initiation
- Alternative promoters allow genes to be transcribed at more than one site - used in different tissues
What are three techniques that are involved in researching the regulation of transcription?
- Deletion mapping
- DNA footprinting
- Genome wide location analysis
What is deletion mapping?
Deletion of a part of a genes promoter to determine what triggers transcription
What is DNA footprinting?
- A transcription factor binding to DNA protects it from enzyme digestion. chromatin treated with DNA digesting enzymes will destroy everything except where the TF bound DNA. Removal of the TF and sequencing
What is genome wide location analysis?
- tool for identifying protein DNA interaction sites on a genomic scale. Chip seq is one method
What is ChIP?
Chromatin immunoprecipitation is an example of genome wide location analysis that allows simultaneous monitoring of all the sites within the genome that bind a given transcription factor.
What are enhancers?
DNA elements that stimulate transcription
What are insulators?
A sequence that can isolate a promoter and its enhancers
What do coactivators do?
Can alter chromatin structure - modifying histones to regulate transcription
How do coactivators alter chromatin structure?
- acetyl groups are added to lysine residues on core histones by histone acetyltransferases (HATs)
- adding acetyl groups to histones tends to increase access to DNA template and promotes transcriptional activation
- histone modifications can show transcribed chromatin regions
- chromatin remodeling complexes use energy from ATP to alter nucleosome structure and location along DNA
What do chromatin remodeling complexes do?
use energy from ATP hydrolysis to alter nucleosome structure and location along the DNA
- are recruited to specific promoters by either epigenetic marks present on nucleosomal histones or other proteins bound to the DNA
What can happen when histone- DNA interactions are disrupted by chromatin remodeling complexes?
- promote mobility of histone so it slides to a new position
- change conformation of nucleosome
- facilitate the replacement within the histone octamer of a standard core histone by a histone variant correlated with active transcription
- remove histone from DNA entirely
What are HDACS? And what does it repress
Histone deacetylases - remove acetyl groups and repress transcription
- subunits of larger complexes acting as corepressors
- recruited to specific gene loci by TF and cause gene silencing
-
What can DNA methylation do>
- can silence transcription in eukaryotes
What are imprinted genes?
genes whose activity depends on wether they originated with the sperm or the egg
- active and inactive versions will differ in their methylation patterns
What are long noncoding RNAs involved in?
- involved in genomic imprinting and X chromosome inactivation - most are associated with gene repression
What four parts are contained in the nucleus?
- chromosomes as extended fibers of chromatin
- nucleoli for rRNA synthesis
- nucleoplasm as the fluid where solutes are dissolved
- nuclear matrix which is the protein containing fibrillar network
What is the structure of the nuclear envelope?
two membranes separated by a nuclear space
- membranes are fused at sites forming nuclear pores
- inner surface of nuclear envelope is lined by nuclear lamina
- contains 60 transmembrane proteins
What is the nuclear lamina?
inner lining of nuclear envelope
- supports envelope
- composed of lamins
- strength regulated by phosphylation of intermediate filaments
What are nuclear pores?
- gateways through nuclear envelope barrier that separates nucleus and cytoplasm
- things can move thru them
What is the nuclear pore complex?
- large doughnut shaped structure that straddles envelope
- composed of 30 proteins called nucleoporins
- huge complex
- NPC is not static- will quickly replace its proteins
What are the proteins in the NPC called?
nucleoporins
What targets cytoplasmic proteins to the nucleus?
nuclear localization signal
What are two transport receptors in the nuclear pore complex?
importins - move molecules from cytoplasm to nucleus
exportins - move molecules from nucleus to cytoplasm
In what form do RNAs move through the NPC?
move as ribonucleoproteins
What is required for mRNAs to be capable of nuclear export?
mRNA must be fully mature - as an mRNA with an unspliced intron is retained in the nucleus
___ contain nucleotide sequences used by the cell to mediate translational control
UTR - untranslated regions
How is the initiation of translation controlled by iron?
- Iron concentrations are low? - iron regulatory protein (IRP) binds to iron response element (IRE) - prevents translation
- iron concentrations are high? - Iron binds to IRP - causes it to dissociate from IRE - allowing translation of mRNA to ferritin
Give two examples of localized mRNA in fly oocytes and what they are precursors to
bicoid mRNA - localized in anterior end cytoplasm - develops head and thorax
oskar mRNA - localized in posterior end cytoplasm - required for formation of germ cells
What determines the lifetime of an mRNA?
poly a tail - starts at 200 A in the nucleus but will get reduced to around 30 A in the cytoplasm
What are P bodies and what can they do?
deadenylation, decapping and degradation occur within p bodies
- can also store mRNA for later translation
What type of RNA is effective in stopping gene expression? What is this known as?
Double stranded RNA, known as RNA interference (RNAi)
what are miRNAs responsible for in development?
- regulate gene expression
- regulatory roles in development
- control of cell proliferation and death
- leaf and flower development in plants
How do miRNAs suppress gene expression?
- act by binding to site in 3 UTR of target mRNA
- promote deadenylation and degradation
- inhibits the initiation of translation
-inhibits elongation - possible activates degradation of nascent peptides
Where is the degredation of proteins carried out?
proteasomes
How do proteasomes function?
- proteasome recognizes proteins linked to ubiquitin
- ubiquitin is transferred by ubiquitin ligases to proteins being degraded
- only polyubiquinated a protein is recognized by the cap of the proteasome
- once degraded the component amino acids are released back into the cytosol
DNA replication is often termed as being ____
semi conservative - each daughter duplex contains one strand from the parent structure
DNA replication starts at the ___ site
origin
What happens at the oriC sequence?
sequence where a number of proteins bind to initiate DNA replication
DNA replication proceeds in what direction in bacterial cells?
bidirectionally
What happens at replication forks?
- parental ds DNA helix is unwinding
- nucleotides are being incorporated into the newly synthesized strands
Topological constraints are an issue for prokaryotic DNA because of the circular shape - what helps with this strain?
DNA gyrase - Type II topoisomerase helps relieve the stress of the supercoiling
DNA synthesis occurs in the _____ direction
5’ - 3’
______ is responsible for synthesizing new DNA strands from a DNA template
DNA polymerase
DNA polymerase requires a ___ to start working
primer
Why is DNA replication in bacterial cells considered semidiscontinuous replication?
Both daughter strands are synthesized simultaneously
- DNA polymerase moves along template in a 3’-5’
- the leading strand is synthesized continuously
- the lagging strand is synthesized discontinuously
What does primase do?
- is an RNA polymerase that assembles short RNA primers
What does helicase do?
unwinds parental strands using ATP
What is DNA polymerase III made of? what is its main function?
- contains 3 core polymerases, various subunits
- synthesizes successive fragments of the lagging strand
- major replicative polymerase - synthesizes leading and lagging strand
What does DNA polymerase I do?
- involved in DNA repair
- removes RNA primers and replaces them with DNA
- mismatch repair mechanisms
Eukaryotes replicate their genome in small portions called ___
replicons
Explain the functions of all 5 polymerases in eukaryotic DNA replication forks
y - replicates mtDNA
B - involved in DNA repair
a - initiates Okazaki fragment synthesis
d - lagging strand synthesis
e - leading strand synthesis
Where does prokaryotic DNA replication take place? Eukaryotic?
prokaryotes - cytoplasm
eukaryotes - nucleus
How many origins of replications are in prokaryotes? Eukaryotes?
prokaryotes - one origin
eukaryotes - many origins
What are the main DNA polymerases responsible for replication in pro and eukaryotes?
pro - DNA pol I, III
euk- DNA pol a, d, e
What is the size difference of Okazaki fragments in prokaryotes and eukaryotes?
prokaryotes - 1000-2000 nt
eukarytoes - 100-200 nt
How fast is DNA replication in prokaryotes and eukaryotes?
prokaryotes - fast, 2000 nt per second
eukaryotes - slow, 100 nt per seconsd
What are three major DNA repair mechanisms?
- nucleotide excision repair
- base excision repair
- mismatch repair
What is nucleotide excision repair?
- cut and patch mechanism
- removes pyrimidine dimers and nucleotides with various chemical groups
What is base excision repair?
- remove altered nucleotides generated by reactive chemicals present in the diet or produced by metabolism
What is mismatch repair?
mismatched bases that are incorporated by the DNA polymerase and escape the exonuclease proofreading
DNA replication takes place during what phase of the cell cyle?
S phase - synthesis
What are three general categories of cells based on ability to grow and divide?
- cells that are highly specialized and lack the ability to divide - nerve cells , muscle cells, red blood cells
- cells that normall do not divide but can be induced to being DNA synthesis and divide when given an appropriate stimulus like liver cells and lymphocytes
- cells that normally possess a relatively high level of mitotic activity, stem cells for blood elements, skin, epithelia
stem cells have _____ cell division in which the daughter cells have different fates
asymmetric
entry into the M phase of the cell cycle is triggered by______
the activation of a protein kinase maturation promoting factor (MPF)
- MPF has two subunits - regulatory kinase and a cyclin
What are the two main points where the cell cycle is controlled?
- START - cell committed to DNA replication (G1 / S cyclins)
- G2 / M transition (mitotic cyclins)
How does cyclin binding facilitate cell replication?
- when a cyclin reaches a sufficient concentration in the cell it binds to the catalytic subunit of a Cdk, causing a major change in the conformation of the enzymes active site
- cyclin binding causes the movement of a flexible look of the Cdk polypeptide chain away from the opening of the active site allowing the Cdk to phosphorylate its protein substrates
Give an example of a Cdk inhibitor in yeast
Sic1 protein acts as a Cdk inhibitor during G1
What is subcellular localization in the cell cycle?
phenomenon where cell cyle regulators are moved into different compartments at different stages
- cyclin B1 shuttles between nucleus and cytoplasm until G2 when it accumulates in the nucleus
probably should go back and look at alllllllll the cdk shit
yuh
Name the stages of mitosis in order
prophase, prometaphase, metaphase, anaphase, telophase, cytokinesis
Define chromatid
one of the two identical halves of a chromosome that hsa been replicated in preparation for cell division
Define kinetochore
large structure that mediates interactions between chromosomal DNA and spindle microtubule polymers
What is the main outcome of prophase?
formation of the mitotic chromosome
what happens during prophase?
- duplicated chromosomes are prepared for segregation
- mitotic machinery is assembled
- chromosome compaction / condensation occurs
-compact chromosomes appear as rod like structures
-
What is condensin? when is it relevant?
protein responsible for compaction
- relevant during prophase
each mitotic chromosome consists of two _____
chromatids
prior to replication the DNA of each chromosome is associated with ____ which forms a ring to encircle the two sister DNA molecules (prophase)
cohesin
Cohesin and condensin both contain ___. What do these do?
both contain SMC proteins - structural maintenance of chromosomes
- SMC proteins fold back on themselves to form a highly elongated anti parallel coiled coil with a globular domain at both termini
Centromeres occur at a ___ ____ on chromosomes and serve as the binding site for ____
primary constriction, proteins
What do kinetochores do?
sites where chromosomes attach to the microtubules of the mitotic spindle
- also the residence of several motor proteins involved in chromosome motility
- on the outer surface of the centromeres
What does the outer kinetochore do? the inner kinetochore?
Outer - microtubule binding
- microtubule motor activity
- signal transduction
Inner - centromere replication
- chromatin interface
- kinetochore formation
During mitosis ____ undergo disassembly before reassembly into the ____
microtubules, mitotic spindle
the centrosome cycle progresses together with what other cycle?
cell cycle
How many centrosomes and centrioles are normally responsible for the formation of the mitotic spindle?
usually a single centrosome and two centrioles, but at the beginning of mitosis the centrosome will split into two adjacent centrosomes
In the mitotic spindles of animal cells, microtubules are arranged in an ____ around each centrosome
aster
What major events occur between prophase and prometaphase?
- dissolution of nuclear envelope and partitioning of cytoplasmic organelles
- basically disassembles most other structures in the cell
What happens in prometaphase?
- mitotic spindle is formed
- chromosomes are moved by microtubules to center of cell
- kinetochore contact stabilizes microtubules
- a single kinetochore is attached to microtubules form both spindle poles
What is the main outcome of metaphase?
- chromosomes are aligned at the spindle equator on the metaphase plate
what are the three microtubule groups in metaphase?
- astral microtubules - radiate from centrosomes to regions outside the spindle body
- chromosomal microtubules -move chromosomes to poles
- polar microtubules - maintain the integrity of the spindle
microtubule flux in the metaphase spindle involves tubulin ____ towards the poles
treadmilling
- subunits are incorporated at the kinetochores of the chromosomal microtubules and the equatorial ends of the polar microtubules
- lost from the minus ends of the microtubules in the region of the poles
What are the two complexes that can add ubiquitin to proteins at different stages of the cell cycle?
SCF - acts during interphase
APC - anaphase promoting complex, regulates events during mitosis depending on the adaptor protein present
What can APC accomplish during anaphase? explain this pathway
- destruction of securin releases an active protease separase which cleaves the cohesin molecule that holds sister chromatids together
- APC(cdh1) completes the ubiquination of cyclin B
- causes a drop in activity of the mitotic cdk and progression from mitosis into the g1 phase of next cell cycle
Why is protein degradation important in regulating the events of mitosis?
- allows cell to reenter G1
- if there is no destruction of cyclin B then cells remain in a late stage of mitosis
What happens in anaphase A vs B
A - movement of chromosomes towards the poles
B - two spindle poles move in opposite directions due to elongation of microtubules
What happens in telophase?
- daughter cells return to interphase
- mitotic spindle disassembles
- nuclear envelope of two nuclei are reassembled
- chromosomes become dispersed
What is cytokinesis?
physical process where one cell is divided into two daughter cells
What does the contractile ring theory suggest?
that a thin band of actin and myosin filaments generate the force needed to cleave the cell
What actually happens for cytokinesis to be successful?
- bipolar myosin filaments composed of myosin II are needed for ring contraction
- force generation is similar to actin myosin
- sliding filaments of the contractile ring pull the cortex and attach plasma membrane towards the center of the cell
the cleavage furrow position is determined by ____
the anaphase mitotic spindle
Define autocrine, paracrine and endocrine
- autocrine - the cell has receptors on its surface that respond to the messenger
- paracrine - messenger molecules travel short distances through extracellular space
- endocrine - messenger molecules reach their target cells through the bloodstream
What is meant by a second messenger?
- small intracellular mediators, small signalling molecules that is formed or released for action in response to an extracellular signal and helps to relay the signal in the cell
At what cell stage do spindle fibres attach to the centromeres of each chromosome?
prometaphase
What do kinases do? what do phosphatases do?
kinases add a phosphate group, phosphatases remove a phosphate group
Provide examples of some extracellular messengers
- small molecules such as amino acids and their neurotransmitter dervitives
- NO and CO
- steroids derived from cholesterol
- eicosanoids, derived from arachidonic acid
- various peptides and proteins
What are the three main receptor types in cells?
- ligand gated channels (ion channel coupled receptors)
- G protein coupled receptors (GPCRs)
- Enzyme coupled receptors (receptor protein tyrosine kinases (RTKs))
A receptor and some of the intracellular signaling proteins it activates in sequence are preassembled into a _____
signaling complex on the inactive receptor by a large scaffold protein
some proteins bind to the ___ _____ whereas some bind directly to the ___ ___
inactive scaffold protein
activated receptor
G protein coupled receptors consist of ___ ____ ______ domains
seven ahelical transmembrane domains
What are the three main methods of extracellular messengers binding to their receptors?
- inactive intracellular signaling proteins bind to scaffold protein
- activated intracellular signaling proteins bind to an activated receptor
- activating receptor causes increases phosphorylation of phosphoinositide’s which act as docking sites for activated signaling proteins
Explain signal transduction by G protein coupled receptors
- ligand binding to the receptor extracellular domain altering conformation
- a GDP is exchanged for a GTP on the Ga subunit, activating it
- Ga binds to effector
- cAMP produced as second messenger
Ga subunits can turn themselves off by hydrolysis of ____
GTP to GDP and Pi
Heterotrimeric G proteins function as ______ and while active, Ga subunits can turn on _________
- molecular timers
- downstream effectors
Heterotrimeric G proteins come in four varieties:
- Gs - couple receptors to adenylyl cyclase
- Gq - contain Ga subunits that activate PLCBeta
- Gi - inhibit adenylyl cyclase
- G12/13 we have no clue
Phosphorylation of GPCRs by GPCR kinase sets the stage for what to bind? what happens upon binding?
- for the binding of arrestins, upon arrestin binding GPCRs become desensitized
The synthesis of cAMP follows the binding of a ______
first messenger, hormone or other ligand to a receptor at the outer surface of the cell
Upon arrestin binding, GPCRs become _____
desensitized
Some phospholipids of cell membranes are converted into second messengers by _____
phospholipases
Protein-tyrosine kinases do what
phosphorylate tyrosine residues on proteins
Phosphorylated tyrosines bind effector proteins that have either a ____ domain or a ____ domain
SH2, PTB
Signal transduction by RTKs is usually terminated by internalization of the _____ through ____
receptor, clathrin mediated endocytosis
What are the three fates of internalized RTKs?
- degraded in lysosomes
- returned to PM
- become part of endosomal signallng compleses and engage in continued intracellular signalling
