Exam 4 ;) Flashcards
Transcription
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
transcription occurs in three steps
initiation
elongation
termination
Intiation
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)
Elongation
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?
Termination
DNA sequence indicates end of the process Transcription ends: RNA polymerase is released RNA molecule is released May be influenced by many factors
Pre-mRNA
primary (first) mRNA transcript, that requires processing before it moves out of the nucleus
Exons
(expressed regions): region of pre-mRNA that remains in the mature mRNA
Introns
(interveining regions): those regions of the pre-mRNA that are not part of the mature mRNA
Pre-mRNA Processing
(making mature mRNA)
cutting introns out
splicing exons together
Prokaryotes transcription
Most of the genomic DNA is coding
mRNA is instantly made into mature mRNA
Eukaryotic Gene Processing
Prior to translation
RNA splicing
Addition of 5’ cap
Addition of poly A tail (3’)
RNA Splicing
removal of introns
snRNPs (small nuclear ribonucleoprotein particles
bind to consensus sequences of pre-mRNA
One binds near 5’ exon-intron boundary
One binds near 3’ exon-intron boundary
Proteins form spliceosome (RNA-protein complex)
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)
Addition of a 5’ cap (G cap)
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)
Addition of a poly A tail
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
Translation
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
Structure / Function of the tRNA
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)
Charging” of the tRNA
Aminoacyl-tRNA synthetases – family of 20 enzymes that required for attachment amino acids to tRNA
Each enzyme specific for one amino acid / tRNA group
Ribosome
site of translation
3 binding sites for tRNA
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
Translation
initiation, elongation
Methionine
charged tRNA binds to AUG start codon
Steps of Translation
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)
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
Polyribosomes
purpose: increase rate of protein synthesis
groups of ribosomes on the same mRNA
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)
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
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
Proteolysis
cutting of a polypeptide chain
Glycosylation
addition of carbohydrates to proteins (glycoproteins)
Phosphorylation
addition of phosphate groups (protein kinases)
Constitutive Genes
expressed at all times
Inducible Genes
expressed only when needed
Receptor-Ligand Binding
Signal transduction
Gene activation vs. gene repression
Cell Cycle
Cyclins (bind CDKs and activate them, progression through the cell cycle)
Expression of cyclin genes at specific points during the cell cycle
Virus-infected Cells
“hijack” host gene expression machinery
Divert it to viral gene expression
When is gene expression regulated?
receptor-ligand binding
cell cycle
virus-infected cells
Where is gene expression regulated?
Transcriptional
Post-transcriptional
Translational
Post-translational
Gene expression is very precisely…
regulated
Transcriptional Regulation
Selective Gene Transcription
(Transcription Factors (TFs)
Repressors: (negative regulation) prevent transcription
Activators: (positive regulation) stimulate transcription
Repressors
negative regulation
prevent transcription
Activators
positive regulation
stimulate transcription
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
bacteriophage
bacterial virus
DNA or RNA genome
May have lysogenic phase
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
Regulation of Translation
miRNA
Modification of the 5’cap
Translational repressor proteins
miRNA
inhibition of translation
Translational repressor proteins
Bind mRNAs and prevent attachment to ribosome
Proteosome
large protein complex that hydrolyzes target proteins
Ubiquitin
76 amino acid protein that targets other proteins for degradation
Alternative Splicing
generation of families of different proteins with different activities and functions from a single gene
Inducible operon
turned off unless needed
Repressible operon
turned on unless not needed
Lac Operon
encodes lactose-metabolizing enzymes
Structure: 3 enzyme genes, promoter, operator
High rate of mRNA synthesis, when needed
No transcription, when not needed
Prokaryotic Gene Regulation
Normally: only make the necessary proteins (conserve energy and resources)
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
Lacatose is a
β-galactoside (a disaccharide with galactose and glucose)
Lactose Metabolism in E. coli
Immediately begin making enzymes (3000/cell in 10min.):
β-galactoside permease
β-galactosidase
Β-galactoside transacetylase
No Lactose Metabolism in E. coli
Low level (few molecules / cell):
β-galactoside permease
β-galactosidase
Β-galactoside transacetylase
Operon
Cluster of genes with a single promoter that are transcribed together
Two types of operons
Inducible
repressible
Inducible Operon
turned off unless neede
Repressible Operon
Turned on unless not needed
Lac Operon (-)
(-) lactose
an inducible system
without lactose repressor is bound to operator sequence
Lac Operon (+)
(+) lactose
presence of lactose
trp operon
a repressible system
Two types of chromatin
Euchromatin: loosely packed, undergoing transcription
Heterochromatin: tightly packed, not actively transcribed
Euchromatin
loosely packed, undergoing transcription
Heterochormatin
tightly packed, not actively transcribed
Regulation of chromatin structure
Histone Acetylation: decrease binding to DNA
Histone Methylation: increase binding to DNA
Histone Acetylation
decrease binding to DNA
Histone Methylation
increase binding to DNA
RNA polymerase binding stie
transcribes protein-coding genes
General TF binding site
generatl transcription factors-bind to promoter site, allowing RNA polymerase 2 to next bind
Gene-Specific TF
binding site
Bound by specific transcription factors
Activators – bind to enhancer sequences
Repressors – bind to silencer sequences
Activators
bind to enhancer sequences
Repressors
bind to silencer sequences
Alternative splicing
Purpose: generation of families of different proteins with different activities and functions from a single gene
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)
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
Post-translational Regulation:protein stability
Ubiquitin – 76 amino acid protein that targets other proteins for degradation
Proteosome – large protein complex that hydrolyzes target proteins
Post-translational regulation
protein stability
Ubiquitin
76 amino acid protein that targets other proteins for degradation
Proteosome
large protein complex that hydrolyzes target proteins
Prokaryotic Genomes
First genome to be sequenced (Haemophilus influenzae) by Craig Venter
Eukaryotic genomes
Are larger than prokaryotic genomes
Have more regulatory sequences (proteins) than prokaryotes
Contain large amounts of noncoding DNA
model organisms
reveal characteristics of eukaryotic genomes
used in the laboratory to determine characteristices that are broadly applicable
Eukaryotic organisms contain
gene families
Gene families
a set of similar genes derived from the same parent gene
Pseudogenes
nonfunctional genes; arise from mutations that cause loss of function (may lack promoter or not splice properly)
Eukaryotic Genomes contain
repetitive sequences
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
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
Retrotransposons
make RNA copies of themselves, copied back into DNA, inserted in new genomic (class 1) locations
DnA transposons
no RNA intermediate and no replication, excised from one location and inserted into another (Class 2)
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
What are the types of sequences in eukaryotic genomes?
Single-copy Genes
Moderately Repetitive Sequences
Highly Repetitive Sequences
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
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
Genomics
study of the genome
Proteomics
study of the proteome
Metabolomics
study of the metabolic intermediates and products an organism produces
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
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
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
Biotechnology
any technological application that uses biological systems, living organisms, or derivatives thereof to make or modify products or processes
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
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 GenesRestriction Enzymes
cuts dsDNA at specific sequences
Making recombinant DNA from DNA fragments:_________
DNA Ligase
Vectors
Carrier for Recombinat DNA
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
Viruses
accommodates many larger eukaryotice genes
infect cells naturally
Expression Vectors
Include the appropriate sequences for transcription and translation
Expression Vectors Prokaryotes
Promoter
Transcriptional termination signal
Sequence for ribosome binding
Expression Vectors Eukaryotes
Promoter (with transcription factor binding sequences) Enhancers Transcriptional termination signal Sequence for ribosome binding Poly A addition sequence
Reporter Genes
used to identify host cells with recombinant DNA
Anitbiotic resistance genes
First: determine cells with plasmid
Second: determine cells with desired insert
Green Fluorescence Protein (GFP)
Emits green light when exposed to UV light
Widely used
Selectable Marker
a gene that can be used to identify cells that contain recombinant DNA
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!
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)
Mutations may be studies in knockout
mice
Homologous Recombination
exchange of segments between 2 DNA molecules, based on sequence similarity; similar sequences align and crossover
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
MicroRNAs
microRNAs
Short, ssRNA that are complementary to target mRNA
Target mRNA degraded
Antisense RNA
Base pairs to mRNA
Partially dsRNA – inhibits translation
Used in anti-cancer therapy (reduced expression of genes associated with cancer)
siRNA
Similar to microRNAs
Short (21-25nt) dsRNA molecules
Discovered in late 1990s
