Exam 4 ;) Flashcards
(129 cards)
1
Q
Transcription
A
generation of RNA from DNA
requires a DNA template
Substrate: Nucleoside triphosphates (ATP, GTP, CTP, UTP)
Enzyme: RNA polymerase
No primer required
Prokaryotes – only one type
Eukaryotes – several types
2
Q
transcription occurs in three steps
A
initiation
elongation
termination
3
Q
Intiation
A
Requires a promoter (DNA sequence to which RNA polymerase binds)
Where RNA polymerase is to bind and which strand of DNA to transcribe
Transcription start site (where transcription begins)
4
Q
Elongation
A
RNA polymerase: unwinds 13 base pairs at a time and reads template strand 3’ to 5’
Adds nucleotides at the 3’ end
Complementary base pairing
Ribonucleoside triphosphates (ATP, UTP, GTP, CTP) joined by phosphodiester bonds, releasing a pyrophosphate
DNA rewinds and RNA made as a single-strand
Proofreading?
5
Q
Termination
A
DNA sequence indicates end of the process Transcription ends: RNA polymerase is released RNA molecule is released May be influenced by many factors
6
Q
Pre-mRNA
A
primary (first) mRNA transcript, that requires processing before it moves out of the nucleus
7
Q
Exons
A
(expressed regions): region of pre-mRNA that remains in the mature mRNA
8
Q
Introns
A
(interveining regions): those regions of the pre-mRNA that are not part of the mature mRNA
9
Q
Pre-mRNA Processing
A
(making mature mRNA)
cutting introns out
splicing exons together
10
Q
Prokaryotes transcription
A
Most of the genomic DNA is coding
mRNA is instantly made into mature mRNA
11
Q
Eukaryotic Gene Processing
A
Prior to translation
RNA splicing
Addition of 5’ cap
Addition of poly A tail (3’)
12
Q
RNA Splicing
A
removal of introns
13
Q
snRNPs (small nuclear ribonucleoprotein particles
A
bind to consensus sequences of pre-mRNA
One binds near 5’ exon-intron boundary
One binds near 3’ exon-intron boundary
14
Q
Proteins form spliceosome (RNA-protein complex)
A
Cuts pre-mRNA at 5’exon-intron boundary Intron forms loop structure Cuts pre-mRNA at the 3’ exon-intron boundary Releases introns (degraded in the nucleus) Joins ends of exons together Result = mature mRNA (exported from the nucleus for later translation)
15
Q
Addition of a 5’ cap (G cap)
A
Modified molecule of GTP
Added to pre-mRNA as it is transcribed
Purpose:
helps mRNA bind to ribosome (preparation for translation)
Protects against digestion (ribonucleases – enzymes which break down RNA)
16
Q
Addition of a poly A tail
A
50-300 adenine nucleotides Added to 3’ end of pre-mRNA Purpose: Helps with export of mRNA from the nucleus Helps with stability of mRNA
17
Q
Translation
A
Conversion of mRNA sequence into the amino acid sequence of a polypeptide (protein)
Change from the nucleic acid “language” into the amino acid “language
20 different amino acids
are encoded by the
nucleic acids
Side chains of amino acids
Unique functions
Increase characteristics
of a polypeptide
18
Q
Structure / Function of the tRNA
A
Amino Acid Attachement Site: Bind / carry particular amino acids (at 3’ end) Anticodon: 3 bases that bind mRNA (noncovalent hydrogen bonds) Interact with ribosomes: 3D structure of tRNA fits surface of ribosome (noncovalent hydrogen bonds)
19
Q
Charging” of the tRNA
A
Aminoacyl-tRNA synthetases – family of 20 enzymes that required for attachment amino acids to tRNA
Each enzyme specific for one amino acid / tRNA group
20
Q
Ribosome
A
site of translation
21
Q
3 binding sites for tRNA
A
A (amino acid) site: region where new tRNA binds to mRNA via anticodon-codon bond
P (polypeptide) site: region where tRNA adds its amino acid to the polypeptide chain
E (exit) site: region where the tRNA (w/o amino acid) briefly resides before leaving the ribosome
22
Q
Translation
A
initiation, elongation
23
Q
Methionine
A
charged tRNA binds to AUG start codon
24
Q
Steps of Translation
A
Codon recognition: anti-codon of tRNA binds to codon at A site
Peptide bond formation: (peptidyl transferase activity of the large subunit)
Elongation: free tRNA is moved to the E site and released; growing polypeptide chain moves to the P site
The process is repeated (until stop codon)
25
Translation: termination
Termination
Stop codon enters the A site (mRNA = UAA, UAG, and UGA)
Release factor binds to complex
Release factor disconnects polypeptide from tRNA in the P site (hydrolysis reaction)
mRNA and ribosomal subunits separate
26
Polyribosomes
purpose: increase rate of protein synthesis
groups of ribosomes on the same mRNA
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Cellular destination of Proteins
Normally:
protein synthesis – begins with free floating ribosomes in cytoplasm…default end-location is cytosol
May contain signal sequence (short sequence of amino acids that indicates cellular location)
28
Some polypeptides are translated into the RER
Polypeptide with 5-10 hydrophobic amino acids at N-terminus --- directed to RER
Polypeptide binds to receptor protein in RER membrane and translation continues
Signal sequence is removed
Translation continues till termination
Ribosome is released and protein folds inside of RER
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Post-translational Modification of Proteins
Purpose: to influence function of the protein
Proteolysis – cutting of a polypeptide chain
Glycosylation – addition of carbohydrates to proteins (glycoproteins)
Phosphorylation – addition of phosphate groups (protein kinases
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Proteolysis
cutting of a polypeptide chain
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Glycosylation
addition of carbohydrates to proteins (glycoproteins)
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Phosphorylation
addition of phosphate groups (protein kinases)
33
Constitutive Genes
expressed at all times
34
Inducible Genes
expressed only when needed
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Receptor-Ligand Binding
Signal transduction
| Gene activation vs. gene repression
36
Cell Cycle
Cyclins (bind CDKs and activate them, progression through the cell cycle)
Expression of cyclin genes at specific points during the cell cycle
37
Virus-infected Cells
“hijack” host gene expression machinery
| Divert it to viral gene expression
38
When is gene expression regulated?
receptor-ligand binding
cell cycle
virus-infected cells
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Where is gene expression regulated?
Transcriptional
Post-transcriptional
Translational
Post-translational
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Gene expression is very precisely...
regulated
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Transcriptional Regulation
Selective Gene Transcription
(Transcription Factors (TFs)
Repressors: (negative regulation) prevent transcription
Activators: (positive regulation) stimulate transcription
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Repressors
negative regulation
| prevent transcription
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Activators
positive regulation
| stimulate transcription
44
Viruses
– regulate gene expression to evade the host immune response
Acellular: depends on living cells to reproduce
Genome: dsDNA, ssDNA, dsRNA, ssRNA
Survival: hijacking host gene expression machinery
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bacteriophage
bacterial virus
DNA or RNA genome
May have lysogenic phase
46
HIV
Causes AIDS (acquired immunodeficiency syndrome
Retrovirus
Enclosed in phospholipid membranes (from previous host)
Membrane proteins: help with fusion of viral PM and infection of host cell
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Regulation of Translation
miRNA
Modification of the 5’cap
Translational repressor proteins
48
miRNA
inhibition of translation
49
Translational repressor proteins
Bind mRNAs and prevent attachment to ribosome
50
Proteosome
large protein complex that hydrolyzes target proteins
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Ubiquitin
76 amino acid protein that targets other proteins for degradation
52
Alternative Splicing
generation of families of different proteins with different activities and functions from a single gene
53
Inducible operon
turned off unless needed
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Repressible operon
turned on unless not needed
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Lac Operon
encodes lactose-metabolizing enzymes
Structure: 3 enzyme genes, promoter, operator
High rate of mRNA synthesis, when needed
No transcription, when not needed
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Prokaryotic Gene Regulation
Normally: only make the necessary proteins (conserve energy and resources)
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3 proteins that are critical for the uptake and metabolism
3 proteins that are critical for the uptake and metabolism:
β-galactoside permease – carrier protein in plasma membrane
β-galactosidase – enzyme that hydrolyzes lactose to glucose and galactose
Β-galactoside transacetylase – transfers acetyl groups from acetyl CoA to certain β-galactosides
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Lacatose is a
β-galactoside (a disaccharide with galactose and glucose)
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Lactose Metabolism in E. coli
Immediately begin making enzymes (3000/cell in 10min.):
β-galactoside permease
β-galactosidase
Β-galactoside transacetylase
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No Lactose Metabolism in E. coli
Low level (few molecules / cell):
β-galactoside permease
β-galactosidase
Β-galactoside transacetylase
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Operon
Cluster of genes with a single promoter that are transcribed together
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Two types of operons
Inducible
| repressible
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Inducible Operon
turned off unless neede
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Repressible Operon
Turned on unless not needed
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Lac Operon (-)
(-) lactose
an inducible system
without lactose repressor is bound to operator sequence
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Lac Operon (+)
(+) lactose
| presence of lactose
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trp operon
a repressible system
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Two types of chromatin
Euchromatin: loosely packed, undergoing transcription
Heterochromatin: tightly packed, not actively transcribed
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Euchromatin
loosely packed, undergoing transcription
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Heterochormatin
tightly packed, not actively transcribed
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Regulation of chromatin structure
Histone Acetylation: decrease binding to DNA
| Histone Methylation: increase binding to DNA
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Histone Acetylation
decrease binding to DNA
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Histone Methylation
increase binding to DNA
74
RNA polymerase binding stie
transcribes protein-coding genes
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General TF binding site
generatl transcription factors-bind to promoter site, allowing RNA polymerase 2 to next bind
76
Gene-Specific TF
| binding site
Bound by specific transcription factors
Activators – bind to enhancer sequences
Repressors – bind to silencer sequences
77
Activators
bind to enhancer sequences
78
Repressors
bind to silencer sequences
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Alternative splicing
Purpose: generation of families of different proteins with different activities and functions from a single gene
80
microRNA (miRNA)
Purpose: degradation of mRNA
Small, noncoding RNAs
22 nucleotides in length
Dozens of mRNA targets
Made as a longer precursor, that is cleaved to make a double-stranded miRNA
First discovered in C. elegans (worm model used to study developmental biology)
81
Regulation of Translation
miRNA – inhibition of translation
Modification of the 5’cap
mRNA capped with unmodified GTP = not translated
Translational repressor proteins
Bind mRNAs and prevent attachment to ribosome
82
Post-translational Regulation:protein stability
Ubiquitin – 76 amino acid protein that targets other proteins for degradation
Proteosome – large protein complex that hydrolyzes target proteins
83
Post-translational regulation
protein stability
84
Ubiquitin
76 amino acid protein that targets other proteins for degradation
85
Proteosome
large protein complex that hydrolyzes target proteins
86
Prokaryotic Genomes
First genome to be sequenced (Haemophilus influenzae) by Craig Venter
87
Eukaryotic genomes
Are larger than prokaryotic genomes
Have more regulatory sequences (proteins) than prokaryotes
Contain large amounts of noncoding DNA
88
model organisms
reveal characteristics of eukaryotic genomes
| used in the laboratory to determine characteristices that are broadly applicable
89
Eukaryotic organisms contain
gene families
90
Gene families
a set of similar genes derived from the same parent gene
91
Pseudogenes
nonfunctional genes; arise from mutations that cause loss of function (may lack promoter or not splice properly)
92
Eukaryotic Genomes contain
repetitive sequences
93
Highly repetitive
Short (less than 100bp), repeated 1000’s of times in tandem
Genome: 10% (humans) to 50% (some species of fruit flies)
Often associated with heterochromatin
Short tandem repeats (STRs):
1-5bps, repeated up to 100X
Chromosomal location varies and is inherited
94
moderately repetitive sequence
Repeated 10 – 1,000 times
Include genes for tRNAs and rRNAs (multiple copies)
Many are transposons…what are these?
40% of human genome
95
Retrotransposons
make RNA copies of themselves, copied back into DNA, inserted in new genomic (class 1) locations
96
DnA transposons
no RNA intermediate and no replication, excised from one location and inserted into another (Class 2)
97
Transposon
sequences of DNA that move around within the genome
Might be inserted into a gene sequence --- alternative mRNA / inactivated gene
Short sequences (1,000 to 2,000bp
98
What are the types of sequences in eukaryotic genomes?
Single-copy Genes
Moderately Repetitive Sequences
Highly Repetitive Sequences
99
The Human Genome Project
Public project completed in 2003
undertaken to determine the normal sequence of the human genome
determine mutations and relate them to phenotypes
100
Overview of the Human Genome Project
3.2 billion bp in haploid genome = 24,000 protein-coding genes
Average gene: 27,000bp (1,000 to 2,400,000bp)
All genes have many introns
3.5% is functional, but noncoding --- role in gene regulation (microRNA)
Over 50% is made of transposons and other repetitive sequences
Most (97%) is the same in all people
101
Genomics
study of the genome
102
Proteomics
study of the proteome
103
Metabolomics
study of the metabolic intermediates and products an organism produces
104
Human Genomics and Benefits for Medicine
Understanding of the genetic basis of disease
| SNPs (single nucleotide polymorphisms) – single nucleotide variations; may vary between individuals or alleles
105
DNA Microarray
may be used to determine which SNPs are associated with human disease (ie. breast cancer, diabetes, arthritis, obesity, and coronary heart disease)
Private companies: scan your genome for SNP alleles
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Pharmacogenomics
study of how an individual’s genome affects their response to drugs
Genetic variations affect how well an individual responds to a particular drug
Analysis used to predict whether or not a person will respond well to a drug
107
Biotechnology
any technological application that uses biological systems, living organisms, or derivatives thereof to make or modify products or processes
108
Recombinant DNA Technology
Single molecule, containing DNA sequences from two or more organisms
```
Four necessary tools:
Restriction enzymes (RE) –
cut DNA into fragments for
manipulation
DNA ligase – joining DNA fragments together
Vector – carrier of recombinant DNA
Reporter Genes
```
109
Recombinant DNA Technology Tools (4)
```
Restriction enzymes (RE) –
cut DNA into fragments for
manipulation
DNA ligase – joining DNA fragments together
Vector – carrier of recombinant DNA
Reporter Genes
```
110
Restriction Enzymes
cuts dsDNA at specific sequences
111
Making recombinant DNA from DNA fragments:_________
DNA Ligase
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Vectors
Carrier for Recombinat DNA
113
Plasmids
Small circular DNA molecules
Autonomous replication within bacteria
Favorable because…
Small (2,000-6,000bp) – easy to manipulate
Contain one or more RE sites – easy insertion of DNA
Contain “resistance genes” – easy selection
Contain bacterial origin of replication – replication independent of host chromosome
114
Viruses
accommodates many larger eukaryotice genes
| infect cells naturally
115
Expression Vectors
Include the appropriate sequences for transcription and translation
116
Expression Vectors Prokaryotes
Promoter
Transcriptional termination signal
Sequence for ribosome binding
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Expression Vectors Eukaryotes
```
Promoter (with transcription factor binding sequences)
Enhancers
Transcriptional termination signal
Sequence for ribosome binding
Poly A addition sequence
```
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Reporter Genes
used to identify host cells with recombinant DNA
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Anitbiotic resistance genes
First: determine cells with plasmid
Second: determine cells with desired insert
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Green Fluorescence Protein (GFP)
Emits green light when exposed to UV light
| Widely used
121
Selectable Marker
a gene that can be used to identify cells that contain recombinant DNA
122
Gel Electophoresis
separation of DNA fragments
The number of fragments:
How many times is the restriction site present in the DNA sample?
The size of fragments:
Use of DNA ladder (for size comparison)
The relative abundance of fragments:
Intensity of band
May use slice of gel with desired DNA for future experiments!
123
DNA mutations can be made in the laboratory
Nature: mutations give cause and effect data
Problem?
Recombinant DNA mutations: more easily studied
Ex. Proteins associated with disease, hemoglobin, NLS (nuclear localization signals)
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Mutations may be studies in knockout
mice
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Homologous Recombination
exchange of segments between 2 DNA molecules, based on sequence similarity; similar sequences align and crossover
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Complementary RNA can be used to
prevent expression of specific genes
purpose: block translation of mRNA
microRNAs
Short, ssRNA that are complementary to target mRNA
Target mRNA degraded
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MicroRNAs
microRNAs
Short, ssRNA that are complementary to target mRNA
Target mRNA degraded
128
Antisense RNA
Base pairs to mRNA
Partially dsRNA – inhibits translation
Used in anti-cancer therapy (reduced expression of genes associated with cancer)
129
siRNA
Similar to microRNAs
Short (21-25nt) dsRNA molecules
Discovered in late 1990s
