General Translation/Proteins
What is Translation and where does it occur?
Describe Initiation in prokaryotes
IF-3 binds to 30s subunit, separating 30s from 50s, which allows mRNA to bind to 30s subunit
Initiator tRNA carries fmet-tRNA^fmet and forms complex with other proteins
This complex binds to AUG start codon on mRNA
Proteins dissociate from complex and 50s binds to 30s creating 70s ribosome which initiates translation
PCR stands for_____________ and its goal is __________________
Polymerase Chain Reaction
Make millions of copies of DNA using exponential amplification
What is the goal of DNA sequencing?
What do restriction enzymes do?
Cleave invading viral DNA (harvested from bacteria)
Serves as scissors that recognize DNA sequence and cuts it-hydrolizes phosphodiester bonds
Goal of agarose gel-electrophoresis?
Separate DNA molecules based on size using agarose
Define Hybridization
The process of producing a hybrid between a labeled probe and a molecule of interest
Define cloning (plasmid cloning)
To make copies of specific DNA fragments onto bacterial vector (plasmid) or other cloning vector
What is a gene library?
COllection of clones DNA fragments that are maintained in a suitable cellular environment
What are the four functions of proteins?
1. Enzymes that enable chemical reactions.
2. Structural components of cells.
3. Transport substances in cells.
4. Regulators in cellular activity.
What is the Shine-Dalgarno sequence and why is it important?
Shine-Dalgarno sequence is an additional binding site consensus sequence on mRNA-8 bp upstream of AUG start site
5'AGGAGGU3'
16s RNA carried by the 30s subunit binds to the SD sequence and anchors the ribosome to the mRNA at the correct AUG initiation site.
Principle is similar to DNA replication and requires the following four 'ingredients'
1. DNA template
2. Taq DNA polymerase
3. Primers
4. Four deoxyribonucleotide triphosphates
Describe the principle of DNA sequencing
dNTP has oh group on C3
ddNTP has h group on C3, terminating replication when incorporated into polynucleotide and marks the presence of that nucleotide by ending replication at that point
The following types of restriction enzymes are involved in producing what?
Blunt ends
sticky 5' ends
sticky 3' ends
Blunt ends- cleaves dsDNA into two dsDNA fragments at the same spot
sticky 5' ends- generates 2 5' overhangs
sticky 3' ends- generates 2 3' sticky ends
Agarose is the polymer of _______ monomer
Agarobiose
What are the three types of hybridization, and what are there goals?
Southern Blot: Detect DNA using DNA probe
Northern Blot: Detect RNA using DNA probe
Western Blot: Detect protein using antibody
The principle of plasmid cloning is the following
Insert DNA into cloning vector by digestion, ligstion, transformation, transformant selection, and blue-white screening
What's the goal of gene libraries?
The goal is to identify gene homologs among species
Polymer of amino acids held together by peptide bonds between an amino and carboxyl end
Protein
Describe elongation in prokaryotes/eukaryotes
fmet-tRNA^fmet binds to p site on ribosome
EF-Tu forms complex with charged tRNA using GTP, which binds to the A site on ribosome
Peptide bond forms between amino acids located on P and A sites, peptide attaches to tRNA on A site, tRNA on p site becomes uncharged
EF-G protein binds to A site and pushes tRNA from A site to P site, and tRNA from P site to E site
EF-G is released, uncharged tRNA exits ribosome and new charged tRNA binds to A site, elongation continues
There are three steps in PCR procedure, what are they?
How many cycles?
Denaturation
Annealing
Extension
25-40 cycles
Requirements in tubes
1. template DNA
2. Primer
3. DNA polymerase
4. 4 types of dNTPs
5. ddNTPS (different one in each tube)
What enzyme reverses the restriction enzyme function?
Ligase
Explain eletrophoresis
DNA is separated by applying an electric field to move negatively charged DNA through agarose
Describe the principle of Southern Blot hybridization
1. Use agarose gel electrophoresis to separate DNA
2. Need filter membrane to transfer DNA from agar to nylon membrane
3. Use DNA probe for hybridizaion
Criteria for appropriate vector
1. Must be able to replicate by itself
2. Must have selection marker- allows you to know introduction of foreign DNA was successful
3. Unique restriction sites
Describe the genomic DNA library approach
1. Isolate genomic DNA from organism
2. Use restriction enzymes to break into fragments
3. Insert each fragment into a vector of its own
4. Transform clones in bacteria
Describe the four levels of protein structure
1. Primary structure- linear sequence of amino acids
2. Secondary structure- polypeptide chain folds and twists producing alpha helix (spiral shaped) or beta sheet (pleated) depending on protein
3. Tertiary- secondary structure interacts and folds further
4. Quaternary- (only some proteins) two or more polypeptide chain interaction
Describe termination in prokaryotes/eukaryotes
When ribosomes encounter termination sequence on mRNA (UAA, UGA, UAG) tRNA rcognizes codons
RF-1 binds to termination sequence on A site which promotes cleavage between polypeptide and tRNA
Polypeptide is released, translation terminates, mRNA degrades
Describe denaturation
Denaturation turns dsDNA into ssDNA, this is achieved using high heat 95C
What do sequencing reactions produce?
How how these products sorted?
The reactions in each test tube produce varied lengths of newly synthesized DNA.
The varied lengths are separated using polyacrylamide gel electrophoresis
Describe approach
mix dsDNA in a tube with restriction enzyme, incubate in water bath
How do you set it up?
What is needed in order to measure DNA fragment sizes?
How do you see the DNA after it's separated?
Fill plasic box with warm agarose mixture making sure to use a comb to make wells, let cool.
Size marker is needed in one well to compare DNA samples
Stain with ethidium bromide (able to intercalate between DNA bases) and use UV light bc Et-Br fluoresces.
Describe the approach of Southern Blot Hybridization
-Run sample thru agarose gel electrophoresis and stain.
-Soak gel at high PH to turn dsDNA into ssDNA
-Transfer DNA from agar to nylon membrane using water to move molecules of DNA from agar to nylon
-add probe solution and let probe bind to complimentary strands for 1 day.
-wash away excess probes and visualize where probe binds on X-ray film
Describe digestion with regards to plasmid cloning
Digestion in plasmid cloning refers to using the SAME restriciton enzyme to cut vector(plasmid) and insert(foreign DNA) into complimentary sticky ends.
After transformation, what is the result?
3 sequenital mRNA nucleotides
Describe translation initiation in Eukaryotes
Formation of an initiation complex- initation factors bind to 40s subunitand separates it from 60s
40s + met-tRNAi^met =complex made using GTP
Complex binds to 5' cap of mRNA and scans for KOZAK sequence -ACCAUGG-
met-tRNAi^met aligns with AUG on mRNA, initiation factors are released and 60s subunit binds to 40s subunit, translation initiates.
Describe denaturation
Denaturation turns dsDNA into ssDNA, this is achieved using high heat 95C
Bands of DNA are now separated, but how can we see them with the naked eye?
X-ray film absorbs the rays emitted by the isotope as it decays, black bands show up on film that correspond to the lengths of fragment DNA
The size of bp recognition site on restriction enzyme controls _______ of cuts.
Frequency
The application of agarose gel electrophoresis
to determine to size, presence, and number of different pieces of DNA
How do you know DNA exists in the genome?
DNA probes hybridize with DNA of interest, showing DNA exists in genome.
Describe ligation with regards to plasmid cloning
Ligation refers to ligating DNA fragments together using ligase.
How do you identify the gene of interest?
Use hybridization with a DNA probe
What are the stop and start codons?
How many codons specify for amino acids?
AUG
UAA, UGA, UAG
60
Describe the mechanism
Bowen-Conradi Syndrome
Healthy individuals have functional EMG1 gene that encodes ribosomal protein and leads to normal translation.
Affected individuals have mutated EMG1 gene that can't produce functional ribosomal protein which leads to poor translation.
Describe annealing
Primers attach to both ssDNA templates in the head to head direction.
Primers flank DNA to be amplified.
This occurs at 55-65C
How do you read the sequence?
Read from top to bottom.
Each line corresponds to a single nucleotide on the complimentary strand 5'-3'
Convert to template strand and rewrite template strand from 5'-3'
4 bp restriciton enzyme cuts at f=
1/256
The goal of SDS-Polyacrylamide gel electrophoresis
separate proteins according to their size
By using the Southern Blot method, we are trying to determine....
If a gene is present in a genome, where the gene is, and whether or not it has one or two copies (1 or 2 bands)
What does ligation produce?
What are we interested in?
We are interested in recombinant plasmids because they have foreign DNA insert.
This method allows us the store DNA without degredation because it is kept within live cells.
What are functions of tRNA?
What is the shape of tRNA?
How many tRNAs are there?
1. Binds to particular amino acids.
2. Delivers amino acid to ribosome for protein synthesis.
Single stranded clover leaf shape with 3 loops and 4 double stranded stems
40 tRNAs
What are the 3 types of post translational modifications proteins undergo
1. Cleavage by enzymes to remove one amino acid
2 Cleavage by enzymes to produce mulitple small peptides
3. Chemical modifications by adding phosphate, methyl-group, or carboxyl to proteins.
Describe extension
Taq polymerase attached to primers and synthesizes daughter strand DNA.
This occurs at 72C, optimal temp for Taq polymerase.
Modern labs no longer use isotopes, ________ are used instead.
How do procedures differ?
fluorescent dyes (different color dye per nucleotide)
Reactions occur in single test tube
6bp restriciton enzyme cuts at f=
1/4096
Why can't proteins be separated using agarose?
The agarose has large pore size so proteins can't be separated by size, since they all get through at the same rate.
Northern blot goal, principle, approach and applications are the same as __________, but it targets ____, and doesn't need to be soaked in a high ____ solution.
Southern Blot
RNA
PH
This step results in three different bacteria populations and involves the up take of foreign DNA through the cell membrane
Transformation
Why do we need cDNA libraries?
It's impossible to ligate single strand mRNA into dsDNA plasmids so we need to utilize convert the mRNA into complimentary DNA
The sequence of tRNA that is complimentary to mRNA is called
Anticodon loop
_____ is responsible for adding a phosphate group to an inactive protein which activates it.
Kinase
Denaturation refers to turning ____ into ______ at a ____ temperature
dsDNA into ssDNA at a high temperature
What are the three applications of DNA sequencing?
1. Used to find genes in genome by computer algorithms. Nucleotides that code for proteins vs random nucleotide sequences
2. Predict relationships by use of homologs (evolutionary relationships)
3. Predict functions of genes (domain of proteins)
8bp restriciton enzyme cuts at f=
1/65,536
Polyacrylamide is the polymer of _______
Polyacrylamide separates ________ of various lengths because it has small enough pores.
acrylamide
proteins
Describe the procedure of Western Blot Hybridization
1. Sample is separated usuing SDS-gel electrophoresis
2. Transfer proteins from gel to nylon membrane
3. Use primary antibody to bind to protein of interest and wash excess away.
4. Use secondary antibody to hybridize with primary antibody, wash to get rid of nonspecific binding.
5. Stain with colorimetric reaction, secondary antibody couples with enzyme, add enzyme substrate=color change.
Describe the differences between the three population resulting from transformation, and what population we are interested in.
Cell with recombination plasmid
Cell with self-ligation plasmid
Cell with no plasmid
Interested in recomb. plasmid bc it has foreign DNA
Describe the procedure of cDNA
1. Isolate ss mRNA
2. Reverse transcriptase has two functions
-poly T primer: binds to poly A tail of mRNA
-RNA dependent DNA polymerase
=ss complimentary strand
3. Revers transcriptase uses DNA dependent DNA polymerase which converts ss cDNA into dsDNA
4. Transform clones in bacteria
Define Wobble
Wobble occurs when the 3rd base pair of mRNA(codon) has a loose paring with the 1st base pair of the tRNA(anticodon)
______ is responsible for removing a phosphate group from an active protein, rendering it inactive.
Phosphatase
Renaturation refers to turning ______ into _____ at a _____ temperature.
ssDNA into dsDNA at a low temperature
How is this used to our benefit?
We're able to clone insulin genes that can be replicated in bacteria to harvest insulin for people.
Plasmid and insulin gene are cut with the same restriction enzyme.
Ligase joins plasmid DNA with insulin DNA and it's overexpressed in bacteria to produce lots of insulin.
SDS page refers to the detergent that is used to
1. linearize proteins by destroying hydrophobic regions
2. Impart negative charge to proteins so separation can occur.
What does it mean if there is a colored band on membrane?
Protein exists
Transformation selection refers to what process?
What cells survive and why?
Using antibiotic plate to kill cells with no plasmid.
Cells with plasmids survive because there's a drug resistant marker on the plasmids themselves.
What does transforming clones in bacteria produce?
Different colonies that each contain a different piece of mRNA
Translating the same proteins from different codons is explained by the ______
Wobble
How does adding a phosphate group activate an enzyme?
Adding a negative charge to a protein can make it functional
The conformational change the protein undergoes due to the additional phosphate group can make it functional
What type of DNA (ss or ds) absorbs more UV light, and why?
ssDNA absorbs more UV light because they have exposed aromatic rings(of bases) whereas dsDNA have buried bases
Describe technique
How are proteins viewed with naked eye?
Proteins and SDS are mized and loaded into wells on a vertically placed polyacrylamide box. Electrophoresis is applied which separates proteins by size
A dye called coomassie Brilliant Blue (-) binds to the (+) amino acid in nonspecific way
detecting the activity of an enzyme using color reagent
Blue white screening allows us to separate
blue and white colonies, self-ligation plasmids from recombinant plasmids
What is the application of cDNA libraries?
-distinguishes between exons and introns
-helps to determine what genes are expressed and where they are expressed
Prokaryotic ribosomes(rRNA) are composed of
Eukaryotic ribosomes(rRNA) are composed of
30s and 50s subunits = 70s
40s and 60s subunits= 80s
What is the word used to describe the following
all transcripts in cells
all proteins in cells
all protein interactions
and metabolites in cells
1. Transcriptome
2. Proteome
3. Interactome
4. Metabolome
low temps favor ________
what happens to primer activity?
renaturation
primers bind to non specific sites, amplifying nonspecific DNA, causing multiple bands on PCR gel.
The application for SDS polyacrylamide gel electrophoresis is
establishing protein size, determining sample purity, and the quantity of protein (density)
Modern technology makes hybridization more efficient by the use of a ________
Define this
Microarray- Supporting material (usually glass slide) onto which numerous molecules/fragments are attached in a regular pattern for use in genetic analysis
How does Blue white screening work?
E.coli mutant has Lac Z gene which produces the back end of beta-galactosidase
Lac Z gene on plasmid produces front end of beta-galactosidase
If there's foreign DNA insert, Lac Z gene is no longer producing front end of beta-galactosidase due to phase shift in DNA
x-gal+back end+front end=blue colonies (self-ligation)
x-gal+back end only=white colonies(recombinant plasmid)
How do you find which colony contains gene you're looking for?
Must screen libraries.
Decribe the rRNA structure
Single stranded folded structure with intramolecular baseparing that allows for a stable secondary structure (Ribosome)
How are primers affected?
Denaturation
Primers are less likely to bind to ssDNA, no bands on PCR gel or small amount of DNA amplified.
By using microarray, scientists are able to
Hybridize thousands of genes or proteins at once, increasing efficiency.
Using fluorescence detection and computer analysis
How to screen genomic/cDNA libraries?
Transfer bacteria colonies on nylon membrane
incubate with DNA/RNA probe
Use autoradiograph and x ray film to see where probe binds
this colony contain gene x
Describe tRNA charging
1. Enzyme= aminoacyl-tRNA synthetase
2. attaches amino acid to it's tRNA (20 total)
3. Enzyme has two binding sites, tRNA binding site and amino acid binding site
4. Utilizes ATP to attach amino acid to 3' OH end of tRNA
Limitations of PCR
Requires prior knowledge of target DNA
Contamination problems
Accuracy(Taq does not have proofereading function)
The size of the fragment is limited to 5kbp because there's no beta clamp
Powerful genomic tool to detect DNA< RNA< or protein interactions
Applications of PCR
-crime scene DNA or pathogens in humans
2. Shows genetic variation
-mutation of nucleotides or genetic drift (allele frequency changes)