1
Q
what is cell theory and by who?
A
by matthias schleiden & theodor schwann 1839
-all organisms are composed of one or more cells
-the cell is the smallest unit that has the properties of life
-cells arise only from the growth and division of pre-existing cells
2
Q
for most multicellular organisms all cells contain ______ DNA
A
identical DNA
3
Q
genome vs proteome
A
genome = full collection of DNA sequence
proteome = full collection of proteins that can be expressed by a cell, tissue or organism
4
Q
central dogma of biology
A
DNA -> RNA -> Protein
5
Q
prokaryotes examples
A
bacteria and archaea
6
Q
eukaryote examples
A
protists
fungi
animals
plants
7
Q
prokaryotic vs eukaryotic cells
A
prokaryotic
- smaller, less complex
- less genetic info
- lack membrane bound organelles
- no endomembrane system
- cell wall
eukaryotic
- membrane bound organelles
-compartmentalization
- endomembrane system
8
Q
describe the nucleus
A
- holds genetic material
(nucleolus = region of intensive ribosomal RNA synthesis) - surface bound by TWO phospholipid bilayer membranes
9
Q
nuclear pore complex
A
- small molecules can pass thru nuclear membrane, large molecules (proteins/RNA) cannot move freely so use nuclear pore complex to enter/exit
10
Q
ribosomes
A
- site of protein synthesis
- complex composed of two subunits that attach to messenger RNA
11
Q
free ribosomes
A
- suspended in the cytosol
- build proteins to be used in the cytoplasm
12
Q
bound ribosomes
A
- attach to membranes, mainly RER
- build proteins to be inserted in membranes or exported from the cell
13
Q
endoplasmic reticulum
A
- compartmentalizes cell
- transport of proteins
- tubules and internal spaces
14
Q
RER
A
- abundant in cells that secrete proteins
- secreted proteins are packaged in transport vesicles
15
Q
SER
A
- synthesize lipids
- permits glucose to exit the cell
- detoxifies chemicals
16
Q
golgi apparatus
A
- collect, modify (glycosylation), tag and package proteins from ER into vesicles to be distributed and used at other locations
- enter thru CIS leave via TRANS face
17
Q
vesicles
A
- used for transport
- pinch off from RER and SER
- bud off from golgi
18
Q
lysosomes
A
- membrane bounded sac that digests macromolecules
19
Q
what’s special about the mitochondria and chloroplasts
A
- both have small quantities of DNA that is prokaryotic in nature
- both grow and reproduce like bacteria
20
Q
mitochondria and the two membranes
A
cellular respiration
OUTER smooth membrane
- holds protons (low pH)
- fluid filled space between membranes
INNER highly folded membrane
- cristae (folds) increase SA for proteins that are involved in electron transport and ATP synthesis
21
Q
mitochondrial matrix
A
- citric acid cycles occurs here
- fluid filled space inside inner membrane
- contains DNA and ribosomes
22
Q
chloroplasts
A
perform photosynthesis
- solar -> chemical energy
- found in plants/ protists
- inner and outer membrane
23
Q
thylakoids & thylakoid space
A
- in the stroma
- contains chlorophyll (absorbs light)
thylakoid space = lumen inside thylakoids
24
Q
eukaryotic cells are large and so need extra support, this is carried out by the _____ and describe
A
cytoskeleton
- spatially organize the cell
- positions and transports organelles
- aids cell movement
25
3 types of cytoskeleton structures and describe
1. microtubules (alpha and beta tublin)
- structural support
- form cilia and flagella
- centrioles/ spindle fibers
2. intermediate filaments
- cell structure ( keratins)
3.microfilaments (actin)
- cell movement
- divide cytoplasm
26
Robert Hooke
first to give the name "cells"
27
who was first microbiologist and discovered what
antonie van Leeuwenhoek
-unicellular organisms
28
light microscope key features (5)
- up to 1000x magnification
- resolution 0.2 um
- must be thin specimens / can add dye to see better
- can look at live specimens
- widely accessible, low cost
29
light microscope limitations
- only 0.2 um resolution
- specimen preparation is required
30
fluorescence microscope features
- 20 nm resolution
- up to 1000x magnification
- can be live or dead CELLS
31
how does a fluorescence microscope work (3)
- light hits a specimen that is labelled with fluorescent dyes (fluorophores)
- the light is absorbed by the specimen and emitted
- light then goes thru a filter that only allows the wavelength emitted when the dye fluoresces (what you see)
32
what is fluorescence
molecules that absorb light at one wavelength and emit it at another (emitted light is always of lower energy)
33
how do fluorescence label a specimen (2) and describe
1. label with a fluorophore directly or indirectly (thru antibodies/ much more common)
2. fluorescent protein is expressed with protein
- there's colocalization and gene fusion of the protein and fluorescent
34
what is immunofluorescence? and it is ____ specific?
fluorophores attached to antibodies which bind to target - more specific
35
what is GFP
green fluorescent protein
-absorbs blue light and gives off green
36
what are transgenic organisms
fluorescence is inserted into all genes so you get light up organisms
37
what are fluorescent confocal microscopes
- uses fluorescent dye labelling
- live or dead cells
- 3D image that's more clear
38
fluorescence microscopy limitations
- resolution
- can only see structures you label
- higher cost then light microscopes but less than EMs
39
electron microscope key features (2)
- highest magnification
- highest resolving power ~0.2nm
40
transmission electron microscope
(transecting)
-black and white image of details/organelles
41
scanning electron microscope
SCANS the specimen producing a 3D image b&w image
42
electron microscopy limitations
- high cost
- no live samples
- TEM needs extensive prep
43
which electron microscope has higher mag and resolution and needs _____ prep
transmission electron microscope and needs MORE prep
44
what are purines
large bases - A&G
45
what are pyrimidines
small bases - C&T / U in RNA
46
how are nucleotides bonded vs bases
nucleotide = phosphodiester bonds
bases = hydrogen bonds
47
DNA has _______ replication
semi-conservative
48
pro vs euk cells
pro
-circular, shorter DNA
euk
-longer, linear DNA
-23 pairs = 46 chromosomes
49
what is a gene
segment of DNA that contains instructions for making a particular protein or RNA molecule
50
multicellular eukaryote chromosomes vs bacteria and unicellular
multi- much more non coding DNA
uni etc - very condensed
51
what is chromatin
DNA + associated proteins
52
what is a nucleosome made up of with what charges
histones (+) and DNA (-)
53
1st level of chromatin structure
- DNA is wrapped around histone to form the nucleosome
-8 histone proteins in core (2 of each H2A, H2B, H3 and H4)
54
2nd level of chromatin structure and higher level chromatin structure
histone 1 (H1) binds to the linker DNA and facilitates condensation to 30nm fiber
-bring nucleosomes in contact
higher level: 700nm
55
chromatin structure is ___ static
NOT static as cells alter the structure
56
what is epigenetics and epigenetic modifications are ____
changes to chromatin that influences gene expression (phenotype) but does NOT change the DNA sequence
are heritable
57
what is heterochromatin and has high histone ____
CONDENSED / transcription "genes off" / inactive
-high histone methylation
58
what is euchromatin and has high histone ____
UNCONDENSED / transcription "genes on" / active
-high histone acetylation
59
what are the possible epigenetic modifications (4)
=histone modifications
-acetylation and methylation
=DNA methylation
=gene imprinting
=X chromosome inactivation
60
histone acetylation
= gene ON (increases transcription)
-HAT (histone acetyltransferase) add acetyl group to lysine residues
61
what do acetylated residues do
generate binding sites for other protein complexes
62
what is chromatin remodeling and its complexes
rearrange chromatin from condensed to accessible state for TF's and other binding proteins
complexes = reposition nucleosomes
63
histone methylation
= gene silencing (no expression/transcription)
HMT (histone methyltransferase) adds methyl groups to lysine and arginine residues
64
DNA methylation
DNMT (DNA methyltransferase) transfers methyl groups to DNA
65
what are CpG islands (3)
-found in many promoter regions
-cytosines can be "on" (usually) or "off"
-recruits activator or repressor proteins
66
unmethylated vs methylated CpG islands
unmethyl
- bind activators (prevent methylation)
- genes "on" transcription
methyl
- repressors (MeCPs) recruited to methylated sites and close chromatin
- genes "off" no transcription
67
paternally vs maternally imprinted
paternally - gene from father silenced so maternal allele is expressed
maternally - gene from mother is silenced so paternal allele is expressed
68
can DNA methylation be inherited
yes to daughter cells
69
histone modification inheritance
when the cell replicates histones are evenly spread 50/50 between the new cells
so new chromosomes will have 50% old histones and recruit new ones
70
which has a greater variety of functions DNA or RNA?
RNA
as DNA only has one main function - to hold instructions
71
transcription overview
RNA polymerase unwinds part of helix just ahead of the active site and reads template strand 3'-5' to build an RNA molecule out of complimentary base pairing - growing the chain 5'-3'
72
what are rRNA
(ribosomal RNA) = form the core of the ribosome's structure and catalyze protein synthesis
73
what are tRNA
(transfer RNA) = serve as adaptors between mRNA and amino acids during protein synthesis
74
what are miRNA
(microRNA's) = regulate gene expression
75
what are mRNA
(messenger RNA) = code for proteins
76
what are snRNA
along with proteins, form the spliceosome complex (snRNP) for splicing of primary mRNA transcripts
77
describe prokaryotic transcription: initiation
RNA Pol requires a sigma factor
-this sigma factor allows RNA Pol to recognize and bind to the promoter
bacterial promoter = 2 consensus sequences: -35 sequence and -10 sequence
-located UPSTREAM of the transcription start site
78
prokaryotic transcription: elongation
-once ~10 bases have been incorporated into the newly synthesized RNA molecule, the sigma factor is released
-tether attaching RNA polymerase to promoter is broken so elongation can occur
-elongation occurs until is reaches a terminator sequence
79
prokaryotic transcription: termination
RNA Pol dissociates from DNA and rebinds with sigma factor
-termination sequence gets transcribed
-promoter sequence does NOT
80
direction of transcription is determined by the _____ as it has _____
determined by the promoter as it has polarity
81
which transcription is more complex?
eukaryotic
82
describe the RNA polymerases
RNA pol I - transcribes rRNA
RNA pol II - transcription of most eukaryotic genes
RNA pol III - transcribes tRNA, 5S rRNA and other small RNA's
83
eukaryotic transcription: initiation
- consensus sequence in eukaryotic promoter = TATA box
- need general transcription factors at promoter to help recruit RNA polymerases
- TFIID binding to TATA box causes distortion in DNA and this flags the promoter to recruit other TF's and RNA Poll II
-once all the general TF's and RNA Pol II has assembled at the promoter (forming the transcription initiation complex) elongation can begin
84
once the DNA distorts (due to TFIID binding to TATA) the promoter is flagged and recruits what (5)
TFIIB
TFIIE and TFIIH
TFIIF
RNA Pol II
other accessory proteins
85
eukaryotic transcription: elongation
-TFIIH pries apart the double helix at start site which exposes the template strand of gene
-TFIIH PHOSPHORYLATES the C-terminal tail of RNA Pol II, releasing the polymerase from the initiation complex
-once elongation begins the transcription factors dissociate from the promoter region
-ends at the terminator where RNA Pol II will dissociate and is de-phosphorylated
86
transcription factors general notes (3)
- eukaryotes require GTF's
- GTF must assemble at the promoter before transcription can begin
- GTFs help position RNA Poll II at the promoter , help pull apart 2 strands of DNA and release RNA Pol II from promoter to allow elongation
87
what are the 3 eukaryotic modifications and where are the enzymes for them located
1. 5' end cap (guanine is added to help protect mRNA)
2. 3' poly A tail (help direct export of mRNA)
3. splicing (introns removed, exons spliced together)
located = C-terminal tail of RNA Pol II
88
mRNA splicing occurs at nucleotide ______ _______ located at intron-exon junctions
consensus sequences
89
what are the 3 important sites for splicing
- 5' splice site (5' end of intron)
- branch-point site (A)
- 3' splice site (3' end of intron)
90
how does splicing work (4)
1. branch point A nucleotide attacks 5' splice site and cuts the sugar-phosphate backbone
2. 5' end of intron covalently linked to A
3. the free 3'-OH of the exon then reacts with the start of the next exon sequence, joins the exons
4. lariat released and degraded in nucleus
91
what are the 5 types of snRNP's involved in RNA splicing
U1, U2, U4, U5, U6
92
what is the spliceosome
RNA protein complex that catalyzes the removal of introns from pre-mRNA
93
when does RNA splicing occur and why use it
when = DURING the elongation phase of transcription
why = to produce different proteins from the same gene
94
how many amino acids are in eukaryotes and how many are essential
20 total and 9 are essential
95
the genetic code is ____ and ______
redundant and universal
96
what is a codon
grouping of 3 nucleotides that codes for an amino acid
97
how many codons are there and how many are sense codons?
64 and 61 are sense
98
why is there only one reading frame
because there's the start codon AUG that dictates the beginning of translation
99
eukaryotic translation: transfer RNA (5)
each tRNA binds to both amino acid and mRNA (linking AA to its codon / bridge)
- amino acid at 3' loop
- anticodon: 3 nucleotides long and pairs with the complementary codon in mRNA molecule
- T-loop: attachment to the ribosome
- D-loop: binds to enzyme to make sure anticodon is paired to correct amino acid
100
whats the wobble hypothesis
3rd nucleotide can allow tRNA to bind to more than one codon
101
amino-acyl tRNA sythetases
catalyzes the aminoacylation reaction
aminoacylation: makes sure the correct amino acid is paired with its anti-codon
synthetases: recognize tRNAs with anticodons and covalently link to these tRNAs
102
which way is codon strand read vs anticodon strand
codon is 5'-3'
anticodon 3'-5'
103
how many rRNA in ribosomes do eukaryotes have and what are the subunits
4
subunits: 80s (60 large/ 40 small)
104
what does the small ribosomal subunit do vs large subunit
small = matches tRNA to mRNA
large = catalyzes peptide bond formation
105
how many binding sites are on a ribosome
3
-A site (aminoacyl)
-P site (peptidyl)
-E site (exit)
106
eukaryotic translation: initiation
- initiator tRNA carries amino acid methionine (Met) and initiation factors help with the association with mRNA
- small ribosomal subunit and tRNA-Met will scan the mRNA until it reaches AUG (start codon)
- large ribosomal subunit is recruited and initiation factors are released
- all newly made proteins have Met at N-terminus (5') for now
107
what is the function of peptidyl transferase and which subunit is it located ?
- catalyzes the peptide bond between amino acids / growing the chain
located in large subunit
108
eukaryotic translation: elongation
- tRNA enters the A site and large subunit translocate, moving the tRNA's into the E and P sites
- small subunit translocates 3 nucleotides along the mRNA molecule to line back up with the large subunit and this ejects tRNA from E site (ready for new tRNA)
109
eukaryotic translation: termination
- stop codon enters A site and a release factor binds
- this binding alters the activity of the peptidyl transferase (has nothing to bind with) and so releases H20
- ribosome dissociates
110
whats the proteasome and which one do we specifically look at
proteasome = degrades short lived and misfolded proteins
ubiquitin = if a protein is tagged with ubiquitin its sent to proteasome to get degraded
111
different cell types within the same organism contain the same ____ but differ in both _____ and ______
contain the same DNA but differ in both structure and function
112
most human cells express about ___% of their genes
50%
113
a cell can change the expression of its genes by ..... (2)
- permanently (cell identity) or in response to its environment
114
what techniques are used to investigate gene expression
for proteins - 2D gel
RNA - PCR or microarrays
115
what is the most important type of regulation and why
transcriptional control because its the most energy efficient and ensures nothing is unnecessarily produced
116
what are transcriptional switches
ensures the organism can respond to its environment
-transcriptional regulators bind to regulatory DNA sequences
117
what are regulatory DNA sequences
almost all genes have these and are used to switch the gene on/off upon the binding of transcriptional regulators
118
whats negative vs positive regulation
negative - bound repressor protein prevents transcription
positive - bound activator protein promotes transcription
119
what makes up the operon
promoter, operator and genes
120
what is trpR
gene that codes for the trp repressor (not part of operon)
121
genes in an operon are all transcribed _____ and this allows for ______ regulation
transcribed together and allows for coordinated regulation
122
what is the trp operon
genes that code for tryptophan synthesis enzymes
123
inactive trp repressor vs active trp repressor
inactive trp repressor (default) = low trp in cell, so need to make it
-RNA polymerase binds to promoter and gene is transcribed
active trp repressor = binds to operator so RNA polymerase cant bind to promoter and gene is NOT transcribed
124
describe activators
bind to regulatory site on DNA and with RNA pol help initiate transcription
125
what is the lac operon
has enzymes (LacZ) that are involved in lactose breakdown
126
for the lac operon where does the repressor bind vs the activator
repressor binds to operator
activator binds to CAP binding site
127
when is the lac operon ON vs OFF
ON = when repressor is not bound ( high lactose) and activator is bound ( low glucose)
OFF = when repressor is bound ( low lactose) and activator is not bound ( high glucose)
128
what is combinatorial regulation
multiple transcriptional regulators work together to control activity of a single promoter
129
high levels of glucose and lactose will produce what level of transcription
low levels of transcription
130
high vs low levels of cAMP
high levels = low glucose, cAMP can bind to CAP so transcription is enhanced
low levels = cAMP cannot bind to CAP site so only low transcription rate
131
does eukaryotic DNA contain operons
no each gene can be controlled seperately
132
are general transcription factors in eukaryotes or prokaryotes
GTFs in eukaryotes
133
where do activators and repressors bind in eukaryotes
activators bind to enhancers
repressors bind to silencers
134
which has more transcription regulators pro or euk
eukaryotes
135
transcription initiation complex
-activator protein binds to enhancer
-promoter is far away
- DNA flips upside down and loops around (with spacer DNA)
- activator protein binds to mediator and other GTFs
136
what do all the transcription regulators do
act as a committee making a decision if should be transcribed or not ( some have strong opinions, weak or neutral)
137
what is the gene control region
includes promoter and regulatory sequences to which regulatory proteins bind to control rate of transcription
138
what is the mediator
takes in all the info from the regulators on whether should transcribe or not
139
why is chromatin remodeling needed for gene expression
HAT loosens up histones and opens up chromatin so transcriptional factors, mediator, RNA can bind and initiate transcription
140
what is coordinated expression
at each gene there is combinatory control but they must be multiple genes with the same regulator acting in order to switch them all on at the same time
141
different combinations of transcription regulators lead to ________________
different types of specialized cells
142
what is de-programming
transcription regulators can push differentiated cells into induced pluripotent stem cells (then can be specialized however you want)
143
once a cell has differentiated into a particular cell type it will ____________ and _______
remain differentiated and all its progeny will stay that same cell type
144
epigenetics allows for ___ _____ and what is it
cell memory
= cells ability to remember its identity (which genes are turned on/off) and these patterns inherited
145
the 3 ways cell memory is obtained
1 - positive feedback loop (master transcriptional regulator activates its own gene expression AND then other genes / is inherited
2 - DNA methylation ( template strand keeps a methyl group and maintenance methyltransferase recognizes it and adds a matching methyl group to new strand/ inherited
3 - histone modifications (daughter cells can inherit parental histone code which determines chromatin structure)
146
#2 and 3 of gene regulation
2 = RNA processing control - RNA splicing allows for different forms of proteins to be expressed
3 = mRNA transport and localization control - nucleoporins regulate nuclear export of mRNAs
147
#4 in gene regulation
mRNA degradation control
148
describe miRNAs steps
- mircro RNAs (base pair with mRNAs to target them for destruction)
- dicer
- associate with RISC
- search for a mRNA match to degrade (good or bad match)
- RISC released
149
describe siRNAs
small interfering RNAs = short FOREIGN RNA fragments that base pair with foreign RNAs to target them for degradation *important in cell defense*
- cleaved by dicer
- associate with RISC
- binds and foreign RNA is degraded
- RISC released
150
#5 of gene regulation
translation control
-no repressor on ribosome binding site = protein made
-repressor on binding site = no protein made
151
#6 and 7 of gene regulation
6 - protein degradation control (ubiquitin / tagged proteins are degraded by the proteasome)
7 - protein activity control (post translational modifications / activate or de-active proteins)
152
what does RISC stand for
RNA-induced silencing complex
153
where can heterochromatin be found
centromeres/ telomeres/ inactivated X chromosomes
BIOL 2202 CMB Midterm 1
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