1
Q
Characteristics of Life
A
o Movement
o Metabolism
o Reproduction
o Response to environment
2
Q
Cell Theory
A
- All organisms are composed of one or more cells (Schwann, Schleiden)
- The cell is the structural and functional unit of life (Schwann, Schleiden)
- Cells can arise only by division from a preexisting cell (Virchow)
3
Q
Organic molecules
A
C,H,N,O,P,S: covalently linked molecules)
Spontaneous synthesis of organic molecules probably provided the basic materials (Miller experiment)
4
Q
Molecules for cataly
A
RNA is only molecule able to both catalyze chemical reactions (ribozyme), and self-replicate (nucleotide base pairing)
RNA: likely the first genetic material in an early stage of chemical evolution leading to formation of primitive cells
5
Q
Prokaryotic cells:
A
: lack a nuclear envelope.
6
Q
o Eukaryotic cells
A
: have a nucleus
Genetic material is separated from the cytoplasm
Eukaryotic cells contain a variety of membrane-enclosed organelles within their cytoplasm
Allows for compartmentalization of structure and function
7
Q
Yeasts
A
the simplest eukaryotes (unicellular); more complex than bacteria, smaller and simpler than cells of animals or plants.
8
Q
Epithelial
A
form sheets that cover the surface of the body and line the internal organs.
Specialized for protection, secretion, absorption
9
Q
• Bright-field microscopy
A
o Requires fixation (killing) of cells/tissues, cutting a thin cross section of tissue, and a variety of stains to provide contrast between subcellular organelles in order to visualize.
10
Q
• Phase-contrast and differential interference contrast microscopy
A
: optical systems that convert variations in density or thickness into contrast that can be seen in the final image without staining.
o Allows visualization of live cells
11
Q
• Fluorescence microscopy
A
widely used and very sensitive method to study intracellular distribution of molecules.
o Fluorescent markers, dyes and proteins (eg. green fluorescent protein (GFP)) used to visualize proteins/structures in living cells.
12
Q
• Confocal microscopy
A
specialized form of fluorescent microscopy, allows for focus on a single plane in the specimen.
o Provides a much sharper image
o Multiple images can then be reconstructed into a 3-dimensional image
13
Q
• Transmission electron microscopy
A
passes a beam of electrons through a thinly sliced, fixed specimen to form an image on a fluorescent screen.
14
Q
• Scanning electron microscopy
A
electron beam reflects off sample surface that is coated with metal, providing 3-dimensional surface image
15
Q
• Nucleosides
A
are a nitrogenous base linked to the ribose or deoxyribose sugar
16
Q
• Nucleotides
A
also contain the phosphate group, and are the basic building block of RNA and DNA
17
Q
Nuclear envelope
A
- consists of two phospholipid bilayer membranes, an underlying nuclear lamina (protein framework), and nuclear pore complexes. Separates the contents of the nucleus from the cytoplasm
- The outer membrane is continuous with the endoplasmic reticulum. It is enriched in membrane proteins that bind the cytoskeleton.
- The inner membrane has proteins that bind the nuclear lamina.
18
Q
Nuclear pore complex
A
- selectively control the traffic of polar molecules, ions, and macromolecules through nuclear envelope. Significantly different from typical membrane proteins.
- Are very large and complex structures – 30x the size of a ribosome
- In vertebrates multiple copies of 30 different pore proteins called nucleoporins
- Organized into 8 spokes surrounding a central channel. The spokes are connected to protein rings at both the cytoplasmic and nuclear side
- The assembly is anchored at fusion points between the outer and inner nuclear membranes
19
Q
Nucleolus
A
- the nuclear site of rRNA transcription, rRNA processing and ribosome assembly
- Large numbers of ribosomes (about 10 million per mammalian cell) are needed by the cell, therefore the nucleolus appears dark due to the large amount of transcriptional activity
20
Q
Gregor Mendel
A
deduced the classical principles of genetics based on the results of breeding experiments with peas.
21
Q
The central dogma
A
REPLICATION thenTRANSCRIPTION thenTRANSLATION
22
Q
Codons
A
the basic units of the genetic code
23
Q
Proteomics
A
the large-scale analysis of cell proteins.
A proteome is all the proteins expressed in a given cell
Proteins function by interacting with other proteins in protein complexes and networks.
24
Q
Genomics
A
complete sequence of the human genome
25
Gene
• Structurally, a gene is a segment of DNA within a chromosome that is expressed to yield a functional product (small % of genome, approximately 21,000 genes in humans)
o Most genes encode mRNAs that are subsequently translated into proteins by ribosomes, but some genes also encode regulatory and structural RNAs
26
o Exons
are segments of protein-coding sequence [and 5’ and 3’- untranslated regions (UTRs)].
• Only 10% (on average) of a typical genes sequence is exons (RNA-coding region)
• Barely 1% of the human genome is exons that actually contain the genetic code sequences that encode proteins
27
o Introns (intervening sequences)
(intervening sequences) are segments of non-protein-coding sequences.
Make up the majority of a genes RNA-coding region in most genes of higher eukaryotic organisms (~35% of human genome)
Can also encode functional products (using nested genes), which may either be proteins or non-functional RNAs
• 150 nested genes have been found in the human genome
28
o RNA splicing
the joining of exons in a precursor mRNA molecule
29
micro RNAs
fold into hairpin structures that are cleaved by nucleases (Dicer) and become double stranded. This is recognized by a RNA-induced silencing complex (RISC) which facilitates RNA degradation thereby inhibiting translation
30
long non-coding RNAs
approximately 50,000 have been found (more than the number of protein coding genes) and their expression is tissue specific.
31
satellite DNA
Small sequences organized in tandem arrays that are repeated millions of times in the genome (used for DNA fingerprinting and finding selectable markers for plant breeding)
32
Transposons:
DNA elements which are capable of moving to different sites in the DNA
33
Retrotransposons
transposition is mediated by reverse transcription (sequence is transcribed to RNA then back to DNA then integrated)
34
DNA transposons
elements are copied and reinserted as DNA
35
How do transposons affect the cell?
o Directly contributes to the evolution of a species through evolution of new genes, but it’s not all positive
o Diseases such as hemophilia, cystic fibrosis, muscular dystrophy and inheritable cancers have been linked to retrotransposons
36
Pseudogenes
non-functional gene copies; have been inactivated by gene mutations and are evolutionary relics
37
Chromatin
eukaryotic chromosomal DNA complexed with proteins, typically has about twice as much protein as DNA.
Two main types: Euchromatin, Heterochromatin
38
Euchromatin
decondensed, transcriptionally active interphase chromatin (usually as 10 and 30 nm fibers, or slightly more condensed).
39
Heterochromatin
highly condensed, transcriptionally inactive chromatin, and it contains highly repeated DNA sequences
DNA is 10,000x more condense in metaphase of mitosis than in interphase
40
Nucleosomes
the basic structural units of chromatin and consist of DNA + histones (chromatosomes + linker)
41
Chromatosome
166 bp + histone H1 (a “linker” histone)
42
o Nucleosome core particles
contain 147 base pairs of DNA wrapped around an octamer (8) consisting of two molecules each of histones H2A, H2B, H3, and H4.
43
Histones
small proteins containing a high proportion of the basic (positive) amino acids, arginine and lysine, which helps facilitate binding to the negatively charged DNA sugar-phosphate backbone.
o H1, H2A, H2B, H3, H4
o Very abundant cellular proteins (total mass approx. equal to mass of cellular DNA)
44
Histone modifications
regulate gene transcription.
o Acetylation of specific lysine groups on the amino tails of histone proteins, neutralize the positive charge of those lysines. This relaxes the chromatin structure (converting heterochromatin into euchromatin) and allows for DNA sequences to be accessible for transcription.
45
o Histone acetyltransferases (HAT):
add acetyl groups
46
o Histone deacetylases (HDAC):
remove acetyl groups
47
epigenetic inheritance
“the transmission of information that is not contained within the sequence of DNA to the daughter cells at cell division
48
Centromere
a specialized region of the chromosome that plays a critical role in ensuring the correct distribution of duplicated chromosomes to daughter cells during mitosis.
Centromeres in all eukaryotes studied to date contain a histone variant within nucleosomes known as centromeric histone H3 (CENP-A or centromere protein A)
49
CENP-A
required for assembly of kinetochore
• CENP–A containing nucleosomes are incorporated only into the centromere. This incorporation is directed by the nucleosomes themselves, not DNA.
• Very important for maintaining fidelity of cell division.
50
Kinetochore
a protein structure associated with the centromere, to which microtubules bind (hips of the chromosome)
o Kinetochore proteins act as a molecular motor during mitosis and meiosis; not associated with centromere during interphase
51
• Telomeres
the sequences at the ends of eukaryotic chromosomes.
o Critical role in maintaining stability of linear chromosomes
o Have been linked to aging and cell reproduction
o Maintained by a unique enzyme known as telomerase
52
• DNA polymerases:
: a group of multi-subunit enzymes that catalyze the synthesis of DNA
1. All polymerases synthesize DNA only in the 5’ to 3’ direction (therefore, copy the DNA template in the 3’ to 5’ direction).
2. DNA polymerases can add a new deoxyribonucleotide only to a preformed primer strand (requires a 3’-OH) that is hydrogen-bonded to the template.
53
• Replication fork
the region of DNA synthesis where the parental strands separate, and two new daughter strands elongate.
54
o Accessory proteins to the replication fork:
```
Clamp loading proteins
Sliding clamp proteins
Helicases
Single-stranded DNA-binding proteins
Topoisomerases
```
55
Topoisomerases
enzymes that catalyze the reversible breakage and rejoining of DNA strands to relieve torsion caused by unwinding of DNA.
• As the strands of parental DNA unwind, the DNA ahead of the replication fork is forced to rotate, creating torsion which can cause supercoiling
56
Single-stranded DNA-binding proteins
stabilize the unwound single-stranded template DNA (DNA does not like to be single stranded
57
Helicases
enzymes that catalyze the unwinding of parental DNA (in ATP-dependent manner)
58
Sliding clamp proteins
load and hold polymerase onto template (ex: PCNA)
| • CLP’s and SCP’s are present to help set up and maintain the replication complex
59
Clamp loading proteins
loads clamp proteins onto replication fork at primer (ex: RFC)
60
o Okazaki fragments
are small pieces of newly synthesized DNA that are joined to form an intact new DNA strand in the lagging strand (each Okazaki fragment is 1000-3000 bp in length).
61
• Primase
enzyme that synthesizes short fragments of RNA (3-10 nucleotides) complementary to the lagging strand template at the replication fork (no 3’-OH required)
62
• Exonuclease
enzyme activity that hydrolyzes (degrades) DNA or RNA molecules from their ends
o RNase H in eukaryotes, DNA Polymerase I in E.coli
63
• DNA ligase
enzyme that seals breaks in DNA strands following replication.
64
• Origins of replication (ori
specific genome sequences that act as binding sites for the protein complex that initiates the replication process.
o A sequence in the DNA where the replication fork begins to assemble
o Proceeds in both directions
65
o Origin recognition complex (ORC)
a 6 subunit complex recognizes the core sequence and binds to it to initiate formation of replication bubble by recruiting other proteins such as Helicase
o Replication origins in other eukaryotic cells less well defined at DNA sequence level, and may involve unique aspects of chromatin structure rather than a specific sequence.
66
• Reverse transcriptase
a DNA polymerase that uses an RNA template
67
• Homeobox
part of the gene that encodes for the module of the protein that binds DNA (transcription factors)
68
o DNA sequence element or promoter element
a specific short sequence of DNA base pairs (usually 5-10 bp) within a gene promoter that may be recognized and bound by a protein(s) (transcription factor) that regulates the rate of transcription
69
Cis-acting DNA sequences
(also called promoter and enhancer elements) serve to regulate expression of genes (rate of transcription)
70
o Basal/core promoter
at the site of transcription initiation. Genes transcribed by RNA polymerase II contain core promoter elements, including the TATA box and an initiator (Inr) DNA sequence (others also exist)
71
o Promoter
the DNA region (typically @200 bp) upstream of the transcriptional initiation site. Contains a unique combination of DNA sequence elements, causing variations which give genes properties to be expressed
72
o Enhancers
transcriptional regulatory sequences that can be located at a significant distance from the promoter (enhancers can be either upstream or downstream of the gene [kb away])
73
• Transcription factors
proteins that are required for RNA polymerase II to initiate transcription. Include general and gene specific transcription factors
74
The steps of the formation of the preinitiation process
1. A TATA-binding protein, along with TBP-associated factors may bind to the TATA box.
2. TFIIB subsequently associates with TFIID at the core promoter
3. Next, RNA polymerase II and TFIIF are recruited using TFIIB as a bridge to TFIID and the core promoter
• RNA polymerase II has a C-terminal domain (CTD) that requires phosphorylation prior to the initiation of transcription (C for carboxy)
4. Finally, two additional factors TFIIE and TFIIH complete the formation of the initiation complex.
• TFIIH has both a kinase activity that phosphorylates the CTD and a helicase activity to unwind the DNA. TFIIE is a structural component
75
• Plasmid DNA
a method by to introduce recombinant DNA into a cell.
76
• Reporter gene
a gene that encodes an easily assayed enzyme or other protein (tells whether plasmid insertion actually worked)
77
• Steroid hormone receptors
transcription factors that regulate gene transcription in response to hormones such as estrogen and testosterone
78
Homeodomain proteins
transcription factors that play critical roles in the regulation of gene expression during embryonic development o
Contain a helix-turn-helix DNA binding domain
79
• Mediators
a multiprotein complex that functions as a transcriptional coactivator in all eukaryotes
o The main function of mediator complexes is to transmit signals from the transcription factors to the polymerase
80
• Coactivators
a type of transcriptional coregulator that binds to an activator (a transcription factor) to increase the rate of transcription of a gene or set of genes
81
DNA looping
allows transcription factors bound to a distant enhancer to interact with proteins in the RNA polymerase/Mediator complex at the core promoter
82
role of TFIIH in transcriptional activation
o Release of RNA polymerase from the basal complex to initiate transcription requires the phosphorylation and helicase activities of TFIIH
TFIIH has kinase activity that can phosphorylate a CTD tail.
83
• Eukaryotic transcriptional repressorsors Function by:
Blocking binding of transcriptional activator to DNA sequence element
• Sterically blocks the activator from assessing the promoter (A)
Repressing mediator activity
Interaction with co-repressors, which serve to modify chromatin structure into a non-permissive state (B)
• This blocks the activator from attaching to the mediator
84
o Chromatin remodeling factors can
Slide nucleosomes along DNA
Change the conformation of the nucleosomes
Eject the histones from the DNA (see right panel
85
Histone tail acetylation and deacetylation
o Generally speaking, histone acetylation results in transcriptionally permissive chromatin (relaxed), whereas histone deacetylation gives rise to a non-permissive state (tight)
• DNA methylation
86
• DNA methylation
Prevent the movement of transposons
| Inactivate genes involved in development and differentiation.
87
The steps of mRNA processing
1. 7-methylguanosine cap addition
2. 3’ end formation and poly A tail addition
3. Splicing
88
7-methylguanosine cap addition
* added during the modification of the 5’ end of a transcript. This begins after 20-30 nucleotides have been synthesized.
* It is composed of a GTP which is modified by a methyl group as are the ribose sugars of the first one or two nucleotides
* Purpose: stabilize the mRNA molecule and correctly orient the mRNA during translation
89
Additional roles of 7-methylguanosine cap addition
ribosomal recognition, controls mRNA half-life, protects 5’ end
90
3’ end formation and poly A tail addition
* directed by a specific sequence
* results in termination of transcription and polyadenylation of the 3’ end of the transcript tells polymerase to “fall off” the strand
* Purpose: help regulate translation and mRNA stability.
91
splicing
removal of introns
92
• Alternative splicing
removal of certain exons to creat multiple different, but related, proteins from the same gene.
93
SR proteins
SR (serine-arginine) splicing factors (SR proteins) will bind to exon sequences to direct where a spliceosome will form
• SR proteins will differ in two different cell types for which a specific mRNA is alternatively spliced. Therefore they help determine the final “choice” of exons that will appear in any one cell type for any one alternatively spliced mRNA
94
2. RNA degradation
* rRNAs and tRNAs are very stable
* Eukaryotic mRNAs have variable rates of degradation (half-lives of 30s to 20 hrs)
* Mechanisms include the length of poly A tails, binding of RNA binding proteins and degradation targeted by miRNAs
* Purpose: allows the cell to respond to the environment
95
Forms of rRNA
5.8S, 18S, and 28S
| rare 45S rRNA gene
96
polymerase I
o Transcribes rRNA
97
• Translation: the players
```
MERITT
o Messenger RNA (mRNAs)
o Elongation factors
o Ribosomes
o Initiation factors
o Transfer RNAs (tRNAs)
o Termination factors
```
98
RNA polymerase III
synthesizes
99
Three steps of tRNA processing
a. RNA cleavage
b. covalent addition of CCA (cyt, cyt, ade tail added to the 3’ end) (same on all tRNAs)
c. base modification (RNA editing)
100
Three steps to “charge” a tRNA
1. Activation of amino acid
2. Addition of amino acid to tRNA
3. Proper folding of tRNA which can bring amino acid to the ribosome
101
o Monocistronic
One gene codes for one protein
| Eukaryotic genome
102
o Polycistronic
One gene codes for more than one protein
Prokaryotic genome
Ex: the lac operon
103
• The process of translation
Initiation (binding)
Elongation (addition)
Termination (stop codon)
104
Three compartments of Ribosome
A (aminoacyl) site: “Arrival”
P (peptidyl) site: “Polypeptide” (Pause)
E (exit) site: “Exit”
105
Peptide bond
catalyzed by peptidyl transferase – an RNA based enzyme in the large subunit
106
• Termination of translation
o Step 1: stop signal (codon [UAA]) in A site
o Step 2: releasing factor in A site at the UAA codon
Release factors are proteins that recognize stop codons and terminate translation of mRNA
o Step 3: polypeptide chain released
o Step 4: release factor causes structure to come apart (ribosomal subunits can be reused)
107
• Post-translational modification
```
PLUG
Phosphorylation and small molecule modifications
Lipid addition
Ubiquitylation
Glycosylation
```
108
• Targets of the Cytosolic pathways
nucleus, mitochondria, chloroplasts, peroxisomes
109
• Targets of the Secretory pathways
nuclear membrane, ER, Golgi, secretory vesicles, plasma membrane, endosomes, lysosomes.
110
o Cis face of Golgi
closest to ER (where proteins enter
111
o Trans face of Golgi
furthest from ER
112
Cardiolipin
• Decreases permeability of inner membrane to protons
| o Normal membranes are impermeable but have leakage, cardiolipin reduces leakage
