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235 Cards in this Set

  • Front
  • Back
How many hydrogen bonds are there between adenine and thymine? Between guanine and cytosine?
2, 3
How many base pairs per turn occur in double helix DNA?
10 base pairs per turn
What is the double helix DNA made of?
Two antiparallel complementary strands that are right-handed
What makes up the hydrophobic core of the double helix DNA?
stacked bases (hydrophobic core + van der waals; hydrogen bonds act as the glue)
What makes up the hydrophilic exterior of DNA?
The charged sugar-phosphate groups which are highly solvated by water
Why do we have high concentrations of cations (think about double helix DNA structure)?
The exterior of the double helix carries two negative charges per base pair and so there is electrostatic repulsion that occurs making the strands more likely to separate. The cations help stabilize the helical conformation by shielding the charges of the phosphodiester groups and decrease repulsive forces.
T/F: Heat denaturation is the preferred method rather than alkaline denaturation. Explain your answer!
FALSE!

Alkaline denaturation is used because it prevents damage to DNA by preventing the breakage of phosphodiester bonds (heat denat. breaks these bonds).
What are several physical changes that occur with denaturation?
Increase in buoyant density, decrease in viscosity, changes in ability to rotate polarized light and absorbance of UV light.
At what pH can DNA be denatured at? What happens?
> 11.3; the N--H groups become deprotonated preventing them from forming hydrogen bonds.
Where does the measurement of absorbance of a DNA complex occur at?
260 nm.
T/F: Absorbance is higher in individual bases rather than from stacked bases.
TRUE! As bases begin to stack, you have increased electronic interactions (absorbance is usually reduced by 40%).
What happens when you increase the temperature (think of what it does to the DNA structure as well as absorbance!)?
Stacking interactions decrease gradually with increasing temperature meaning that you have an increase in absorbance now since you disrupted the electronic interactions. The ordered structure is disrupted.
When looking at the denaturation curve, when does complete strand separation occur?
Upper plateau of the curve!
What is Tm?
It is the temperature at which 1/2 of the maximal optical density has been reached.
What is Tm a characteristic of?
It's a characteristic of the base content of DNA under standard conditions of concentration and ionic strength.
What happens to the transition temperature between double-stranded helix and single strands when you have a high GC content?
The transition temperature is also higher.
What is renaturation?
AKA reannealing; complementary DNA strands can reform a double-helix
T/F: The formation of the first base in renaturation is very fast. Explain your answer.
FALSE! It is very slow because it's entropically unfavorable at first. However, after you form the first base pair, especially after the formation of the nucleation site (3-5 bp), renannealing occurs rapidly.
What conditions can A-DNA be found in?
It can be found in high concentrations of salt and low humidity. It's shorter and thicker (11 bp/turn). It is also right-handed.

Side notes:
- low humidity exposes more hydrophobic surface to the solvent
- have consecutive guanines on one strand
- underwound
What conditions can B-DNA be found in?
High humidity and low salt concentration. It is the most common one in cells.

Side notes:
- phosphate groups are more accessible to water molecules than in A-DNA
- base pairs are nearly perpendicular to the helical axis
What is Z-DNA?
A left-handed helix with a zigzagging backbone due to the major groove of B-DNA having "popped out" to form the outer surface of Z-DNA.
T/F: DNA is a straight and uniform structure.
FALSE! The reason why DNA has so many different variations in structure and conformation is that there are many specific DNA sequence motifs.
T/F: Most DNA in bacteria exists as closed circles.
TRUE! This includes chromosomal DNA and and extrachromosomal DNA (plasmids).
Are plamids maintained and replicated with chromosomal DNA?
No! They are maintained and replicated separately!
What two organelles in eukaryotic cells contain circular DNAs?
Mitochondria and chloroplasts. The sizes are typically similar to bacterial chromosome.
What is the biologically active form of DNA?
Superhelical.
The strained isomer, DNA, is created by what two processes?
Overwinding and underwinding.
What is underwinding? What is overwinding?
Underwound DNA = negatively supercoiled and right-handed superhelix

Overwound DNA = positively supercoiled and left-handed superhelix
What is the main purpose of topoisomerases?
Catalyze the breakage and joining of DNA to regulate the formation of superhelices.
What is the function of topoisomerase I? topoisomerase II?
Topo 1: binds to one strand of the DNA and separates the complementary strand. It nicks one of the strands and binds to the new termini. The strand that was not nicked passes through the gap and the enzyme ligates the nicked strand together.

Topo 2: You have two types of supercoils (+ and -). The enzyme breaks the back segment (+ coil) and reseals the break on the front side. Now you have 2 negative helices.
What is a nucleoid composed of?
Circular chromosome is compacted into 40-50 loops of supercoiled DNA and organized by a central scaffoled rich in protein and RNA.
What is the difference between prokaryotic and eukaryotic scaffolds?
Prokaryotic - DNA/RNA interactions.

Eukaryotic - DNA/Protein interactions.
What's the difference between RNase and DNase?
RNase - completely unfolds the chromosome by disrupting the nucleoid core.

DNase relaxes the structure one loop at a time.
What is a nucleosome made out of?
A strand of DNA (146 bp) is wrapped around an octomeric histone (2 molecules each of H2A, H2B, H3, and H4).

It is disk-shaped.
Where are the histones in contact with (think of what part of the DNA structure)?
The minor groove! The major groove is for proteins which are important for gene regulation and DNA function.
How are polynucleosomes formed?
Numerous nucleosomes are linked together by a "linker" DNA (20-90 bp long). This DNA associates with histone H1 which locks the supercoil in its place. And this is called a chromatosome
How many base pairs is the chromatosome composed of?
166 (two superhelical turns)
Explain the organization of eukaryotic chromatin
Naked DNA --> DNA wraps around the octomeric histone --> condensations with H1 to form nucleofilament (10 nm) --> eventually will form a 30 nm fiber --> packaged into a twisted, looped structure and attached to scaffold --> chromosome
What is the structure of RNA?
Contains ribose, uses uracil instead of thymine, generally single-strandded
T/F: RNA is stable.
FALSE! The 2' -OH is present (unlike DNA) and so hydrolysis of RNA is accelerated!
Describe the secondary structure of RNA.
It involves intramolecular base pairing. Helices within RNA are usually A-type.

Side notes:
- double-helical stem-loop regions in RNA form hairpins (variation in the fine structural details)
- can have unpaired loops
- anticodon region is unpaired and forms a loop
Describe the tertiary structure of RNA.
It results from base stacking and hydrogen bonding between different parts of the molecule. It also contains an acceptor stem and anticodon loop.
- folded into a compact "L-shaped' conformation.
What is the function of tRNA?
Transfer RNA: 1. activates amino acids 2. recognizes codons in mRNA.
What is the function of rRNA?
Ribosomal RNA: protein synthesis.
What is the function of mRNA?
Carry the information needed for the primary structure of proteins.
What is a ribozyme?
Enzymes that contain RNA subunits that carry out catalytic reactions.

5 classes: 3 are involved with self-processing reactions while 2 are true catalysts (ribonuclease P and rRNA)
What are the major bases in DNA and RNA?
Purines: A & G
Pyrimidines: T & C & U (RNA)
What is a ribonucleoside?
Ribose + BAse
What position of the ribose interacts with the base?
1'
What is a deoxyribonucleoside?
2-deoxyribose + Base
In 2-deoxyribose, what is lost (think of functional group as well as what position)?
-OH group @ 2'
What is a nucleotide?
phosphate + base + sugar
What position of the pentose interacts with the phosphate group?
5'
What is the name of the phosphate group that is directly connected to the pentose?
alpha phosphate
T/F: When you synthesize DNA, the beta and gamma phosphates remain attached to the alpha phosphate.
FALSE! Alpha phosphate remains but the beta and gamma phosphates are broken and released.
Nucleotides are linked together by ___ to form ___.
3', 5'-phosphodiester bonds; polynucleotides
What are the requirements of DNA replication?
Template: provides sequence information.

Primer: provides the 3'-OH to which you add nucleotides to

Precursor: 5-dNTPs

Enzymes: DNA polymerase, helicase, ligase, nuclease, DNA binding proteins, sliding clamps
What is the function of DNA polymerase?
Catalyzes the addition of nucleotides to the 3'-OH end during elongation. It requires both templates and primers and uses 5'-dNTP precursors.
What are two ways DNA polymerase can ensure accuracy?
1. Initial selection of the nucleotide to be added.

2. Enzymatic proofreading (3' to 5' nucleolytic activity) that removes mispaired nucleotides from the 3' end.
What is the major replication polymerase in E.coli? What is the major replication polymerase and the priming replication polymerase in eukaryotic cells?
polymerase III; polymerase delta, polymerase alpha
What is a leading strand? What is a lagging strand?
Leading strand - the 3'-OH group orients towards the fork. This is where you add the nucleotides during elongation.

Lagging strand - the 5'-OH end is oriented towards the fork
What are Okazaki fragments? Okazaki fragments in human cells contain how many nucleotides? How about in E.coli?
Small pieces from which the lagging strand is made. 130-200 nucleotides. 1000-2000 nucleotides.
What is meant by semiconservative DNA synthesis?
Parental strand separates and serves as a template for synthesis of the new (daughter) strand. The result is a pair of duplexes -- one old and one new.
Explain the replication fork movement.
Initially, helicase separates the parental strands and the SSB prevents the strands from reannealing.

The leading strand is elongated at the 3' end by adding nucleotides.
Primase synthesis RNA primers by using the template of the lagging strand.
DNA polymerase extends the 3' end of the RNA primer by adding deoxyribonucleotides.
As a growing Okazaki fragment approaches a previously synthesized primer, the RNA primer is removed creating a gap via the RNA hybridase/polymerase I.
The gap is filled by a DNA polymerase that elongates the Okazaki fragment.
When the gap has been filled, the nick is sealed by a DNA ligase.
What is the difference between a nick and a gap?
Gap: one nucleotide is missing and is filled with deoxytribonucleotides by a DNA polymerase.

Nick: interruption in the phosphodiester backbone with no missing nucleotides
How are sliding clamps formed? What does it help with?
Clamp-loading protein binds to DNA. The clamp loader assembles the sliding clamp. DNA polymerase associates with the clamp.

Helps increase the speed and accuracy of replication.
What are the main components/enzymes used in eukaryotic replication?
DNA polymerase delta associates with PCNA (sliding clamp). The 3 subunit PCNA is assembled by the RFC (clamp-loading factor).

RPA (single-stranded DNA protein) prevents the separated parental strands from reannealing.

The primase forms a complex with DNA polymerase alpha = pol alpha/primase complex. This primer complex synthesizes 10 ribonucleotides and then switches to DNA polymerase activity to begin elongation.

RNA hybridase removes RNA primer leaving 1 nucleotide.

FEN1 removes the last nucleotide by peeling back one or a few nucleotides to form a small "flap".
T/F: The ORC (origin recognition complex) assembles at multiple ori.
TRUE!

Side note: assembly of an ORC at an origin is necessary but not sufficient for initiation to occur.
What is an MCM?
Minichromosome maintenace protein - has a weak helicase activity, must also bind, forming a pre-replicative complex

Side note: activation of the ORC/MCM complex is regulated by cyclins and cyclin-dependent protein kinases.
Where does replication begin?
It begins at a specific site called the origin of replication. Oris contain multiple, short, and repeated sequences which proteins can bind.

AT-rich regions is the initial site where separation occurs.

E.coli has a single origin of replication = oriC (245 bp).

Thousands of origins in eukaryotic cells. DNA replication occurs during the S-phase.

Yeast = ARS (autonomously replicating sequence).
What is the function of telomerase? What is a telomerase?
Catalyzes the addition of new 6 nucleotide telomere repeats to the 3' end of the DNA chain.

Telomerases are ribonucleoprotein complexes containing a small RNA that acts as a templates to add the new 6 nucleotide repeat.

Telomerase binds to the end of the 3' strand. 6 nucleotide repeat is synthesized using the RNA as a template. Telomerase dissociates.
What are the key features of the Holliday model of homologous recombination?
1. homology

2. symmetry of both breaks and strand invasion.

3. presence of a four-stranded "Holliday junction" as a key intermediate
What is recombination? What is homologous recombination?
Recombination is the exchange of genetic information.

Homologous recombination = occurs between identical or nearly identical sequences.
What is transposition?
Movement of specific pieces of DNA in the genome. It resembles site-specific recombination in being catalyzed by special enzymes.

Transposons have two key features that enable them to move nearly anywhere into a target chromosome.
- encode transposase enzymes and have insertion sequences that are recognized by the transposase.
Sometimes transposons carry other genes with them when they transpose.
Is DNA replication 100% accurate?
NO.
*Side notes about DNA repair.
- DNA in cells is constantly being altered by cellular constituents.

- Many environmental agents attack and modify DNA.

- Thus maintenance of the genetic information requires constant repair of DNA damage.
What are the basic steps of excision repair?
1. Recognize damage
2. Remove damage by excising part of one strand
3. Resynthesize to fill the gap using the genetic info from other strand.
4. Ligate!
What is base excision repair?
Involved in repairs of damaged bases - including methylated, deaminated, and oxidized bases, and abasic sites.

1. Remove single damaged base by DNA glycosylase (doesn't break the sugar-phosphate backbone of the DNA).
2. AP endonuclease cleaves phosphodiester bondat 5' side but leaves the sugar attached to the next nucleotide.
3. AP lyase cuts 3' to remove sugar.
4. The gap is filled by DNA polymerase.
5. DNA ligase seals nick
What is nucleotide excision repair?
Repairs damage typically involving large adducts or distortion of the double-helical structure of the DNA.

1.Double excision removes damage as part of an oligonucleotide (12-13 nucleotides in E.coli; 27-29 nucleotides in humans)
2. DNA polymerase fills gap.
3. DNA ligase seals nick.
What is mismatch repair? How does mismatch repair occur in E.coli?
A specialized form of nucleotide excision repair that removes replication errors. Mismatches are not like DNA damage: no damage/modified base present! Just the wrong one of the four bases. Recognition of mismatches depends on the distortion of the double helical structure. Newly synthesized DNA isn't methylated so it's easier to recognize. Enzymes recognize damaged bases specifically.

E.coli
- MutS creates a loop that contains the mismatch and MutL binds and begins the cleavage and excision. MutH nicks the unmethylated, newly synthesized strand on either side of the mismatch. DNA polymerase fills the gaps and DNA ligase reseals the nick.
How is DNA repair regulated?
DNA damage triggers the SOS response which is regulated by a common repressor, LexA. Damage blocks replication forks so you're left with a single-stranded DNA that RecA can bind to and become activated.

The activated RecA can aid in the cleavage of LexA.

As long as the damage remains and RecA remains activated, LexA continues to be cleaved so the operon remains on. When damaged is repaired, RecA is deactivated and no longer cleaves LexA so now LexA builds up.
What is transcription? What are the three parts to transcription?
Transcription is the process in which you synthesize RNA from a DNA template via the use of RNA polymerase.

Initiation, elongation, and termination.
What happens to the DNA double helix as it's transcribed? What is the enzyme that catalyzes transcription and where does it occur?
As it's transcribed, the DNA helix becomes more unwound and opened. RNA polymerase and it occurs in the nucleus and mitochondrial matrix.

*Side note: nucleosome patterns are disrupted so active chromatin is more accessible to the enzymes.
T/F: RNA polymerase requires a primer.
FALSE!
When looking at the general gene structure for transcription, what are the particular proteins and DNA sequences used? Where are they located (upstream or downstream)?
Proteins: activator, promoter, RNA polymerase, repressor, and transcription factors.

DNA sequeunces: enhancer, silencer, and promoter.
Which way does the promoter move in transcription?
It is located upstream but it moves downstream in transcription.
What does an enhancer do?
Stimulates the transcription of RNA but the enhancer is located further away from the initiation site.

Side note: enhancer must be on the same DNA molecule as the gene.
T/F: An enhancer sequence can only function in one orientation and at one distance.
FALSE! It can function in either orientation and at variable distances
This interaction facilitates transcription by "recruiting" RNA polymerase to form an initiation complex.
binding of an enhancer to an activator
What is a prokaryotic promoter? What are the two short sequences that are highly conserved?
specialized DNA sequences required for transcription initiation and located a short distance upstream from initiation sites.

-35 region TTGACA
-10 region TATAAT
What direction does RNA polymerase synthesize RNA in? What is RNA polymerase made out of?
5' to 3'.

RNA polymerase:
1. Core enzyme (2 alpha, 1 beta, and 1 beta prime subunits) - capable of transcription but not specific RNA synthesis.
2. Holoenzyme (1 sigma factor) - capable of specific RNA synthesis
Explain what occurs during initiation.
RNA polymerase holoenzyme binds to the promoter DNA to form this closed complex since it is weakly bound. Eventually It becomes tightly bound to form a open complex characterized by a local opening of 10 bp of the DNA double helix.
Explain what occurs during elongation.
Binding-bond-formation-translocation cycle -- 40 bp per second.
Explain what occurs during termination.
End and release!
What is a rho-independent terminator?
G-C rich stem and loop followed by a polyU residues.
What is a rho-dependent terminator?
It is less defined and contains sequences of regularly spaced C residues. The newly synthesized RNA wraps around the rho factor while ATP hydrolysis leads to dissociation of the transcript from the template.
What is a rho factor?
Hexameric protein that has ATPase activity.
T/F: Once the RNA polymerase has cleared the promoter region, other RNA polymerases can bind and initiate at the promoter so that the gene can be transcribed by many polymerases at the same time.
TRUE! You can have simultaneous transcription of a gene by many RNA polymerases therefore, increasing length of RNA molecules.
What also aids in the process of unwinding and restoring the DNA double helix during elongation?
DNA topoisomerase I & II
What are the molecular events required for transcription initiation in eukaryotes?
1. Chromatin containing the promoter must be made accessible.

2. Transcription factors must bind to DNA sequence in the promoter region to be active.

3. Enhancers must bind to activator to stimulate transcription.
What is the difference between active chromatin and inactive chromatin? What is DNase I hypersensitivity?
Active chromatin is a looser conformation (lacks nucleosomes).

DNase I hypersensitivity: enhanced accessibility promoter sequences
What are the 2 promoters for mRNA synthesis?
TATA box: centered about 25 bp upstream from transcription unit.

CAAT box: located further upstream but not as conserved as TATA.
What is RNA polymerase II responsible for? Does RNA polymerase II become modified?
Initiates synthesis of mRNA in the nucleus. Yes it becomes modified and recruits other nuclear components (RNA processing enzymes for the elongation phase)
What recruits more RNA polymerase in mRNA transcription?
Bound transcription factors!
T/F: Synthesis of rRNA is rate-limiting for cell growth.
TRUE!
What are the transcriptional units that make up RNA polymerase I?
28S, 18S, 5.8S
What are transcriptional units separated by?
Spacer sequences
What is RNA polymerase I involved in? Where does it occur?
synthesis of mRNA, nucleolus
T/F: Transcription of rRNA can be very rapid.
TRUE! Why? Synthesis of ribosomes is rate-limiting for cell growth and the demand for rRNA can be very high.
___ & ___ are transcribed by RNA polymerase III.
5S RNA and tRNA
What binds to the sequence located within the coding sequences of the gene for 5S rRNA? Is this found in tRNA?
TFIIIA; No, it's not found in tRNA.
T/F: Introns must be removed precisely so that the mRNA can accurately encode a protein.
TRUE!
What does Dicer do?
It is an RNAse III nuclease that cleaves dsRNA and miRNA into siRNA (21 bp long).
After the formation of siRNA, what can happen next?
RISC can cleaves mRNA at the double-stranded region.

miRNP stops translation of the mRNA.
T/F: RNA is generally stable with a long half-life.
FALSE! It's generally unstable with a short half-life.

*Side notes:
- RNA molecules are replaceable.
- RNase (ribonucleasE) remove RNAs from the cytoplasm
- structure of RNA affects nuclease action (ex: highly ordered structure --> RNase is less efficient)
In terms of processing, which end of the mRNA is modified by a cap structure?
5' end
What do the reactions of RNA processing include?
1. Base modification
2. Export of RNA from nucleus
3. Addition of nucleotides
4. Removal of extra nucleotides (introns)
5. Separation of different RNA sequences by nucleases



ACRONYM: BEARS!
What is tRNA modified by? Explain each process.
1. Cleavage: cleavage of extra nucleotide sequences as well as any introns in the anticodon region.
- enzymes: ribonuclease P (ribozyme that cleaves the primary transcript), exonuclease, endonuclease, and ligase

2. Addition: CCA is added to the 3'-end (essential for tRNA to accept amino acids)
- enzyme: tRNA nucleotidyltransferase adds the CCA sequence (*CCA ends are found on both cytosolic and mitochondrial tRNAs)

3. Modification: > 60 modifications requiring > 100 enzymes.
- some reactions are simple such as methylations while others involve multistep synthesis.
T/F: Transfer RNA nucleotides are the most highly modified of all the nucleic acids.
TRUE!
After rRNA processing, how many pieces of rRNA do you get for eukaryotes? Prokaryotes? What are they?
Eukaryotes: 3 pieces; 28S, 5.8S, and 18S

ProkarayoteS: 2 pieces; 23S and 16S
What is the Poly(A) tail signal sequence? What does it do? What is the primer for poly(A) synthesis?
AAUAAA. It specifies the cleavage of mRNA precursor. The primer is the free 3'-OH of the mRNA.


*Side note: RNA polymerase will transcribe until it reaches a polyadenylation signal sequence. C-terminus of the RNA polymerase II recruits cleavage and polyadenylation to the transcript; it also terminates transcription.

Final product: fully functional mRNA with all the introns removed (ready for pr- synthesis!)
What is the function of snRNPs (small nuclear ribonucleoproteins)?
break the introns at the 5' donor site and joining the upstream and downstream exon sequences together.
What do all introns begin and end with? What does U1 RNA and U2 RNA recognize?
begin: GU; end: AG

U1 RNA: donor GU sequences
U2 RNA: acceptor AG sequences
What carries out the accurate removal of an intron?
Spliceosome
Explain the scheme for mRNA splicing.
1. 2'-OH group of intron sequeunce reacts with 5'-PO4 of intron's 5'-terminal nucleotide = 2'-5' linkage

2. Simultaneously, the exon 1-intron phosphodiester bond is broken leaving a free 3'-OH terminus on this exon to interact with 5'-PO4 of exon 2, displacing the intron and creating the spliced mRNA.

3. Cellular nucleases digest the released intron lariat
What can alternate pre-mRNA splicing lead to?
It can lead to multiple proteins being made from a single gene.


Ex: tissue-specific tropomyosin proteins come from a single gene.
Describe eukaryotic mRNA.
- monocistronic.
- 5' end is capped and untranslated region
- 3' end is untranslated sequence
- 100-200 nucleotide long poly(A) tail
- initiation signal for translation = AUG (methionine)
Describe prokaryotic mRNA.
- 5'-end is not capped
- no poly(A) tail
Genetic code uses _____ alphabet of nucleotides. Codons are arranged in ___.
4-letter alphabet; 3-letter words
How many total "words" are there? What is the start codon? What is the stop codon?
64 "words"

START: AUG

END: UAA, UAG, UGA


*Side notes:
- sentence --> gene
- paragraph --> related genes
- novel --> chromosome
T/F: Codon usage is universal.
FALSE! It is not universal in mitochondrial code.

(ex: UGA codes for termination but in terms of mitochondria, it codes for typ).
Define point mutations.
Change in a single base pair in the DNA -- single base in the corresponding mRNA.
Define silent mutations.
A point mutation in the 3rd position of degenerate codon - no change is made to aa or protein being encoded.
Define missense mutations.
Base change that causes incorporation of a different aa in the encoded protein.
Define nonsense mutations.
Formation of a termination codon from one that encodes an aa.
Define "read through" mutations.
Mutation of a STOP codon to one that encodes for aa; translation continues past point until the next STOP codon is encountered.
Define frameshift mutation.
Insertion or deletion of one or two nucleotides; the "reading frame" shifts from that point forward
If 3 nucleotides are inserted or deleted, would it be a frameshift mutation?
NO! Duh, a new codon was added.
What are some characteristics of tRNA?
- acceptor stem contains the CCA sequence at the 3'-OH terminus where aa is bound
- anticodon loop interacts with mRNA to recognize the codon to translate into aa sequence.
- L-shpaed, 3D structure (conserved cloverleaf secondary structure)
What occurs in a codon-anticodon interaction?
You have translation of the codons of mRNA which involves direct with complementary anticodon sequences in tRNA.
T/F: Anticodon-codon base pairing is parallel.
FALSE. It's antiparallel
In order for amino acids to be incorporated into proteins, what must happen to amino acids?
They must become activated by linking them to the correct tRNA carriers.
What are the steps necessary for linking an amino acid to the appropriate tRNA?
2 step process. aminoacyl-tRNA synthetase catalyzes the process (20 different types)

STEP 1: aa + ATP + enzyme = forms complex
STEP 2: then complex "finds" tRNA and moves aa onto it.
In the cytosol of animals, what is the monomer size of ribosomes? How about the small subunits? Large subunits?
monomer size: 80S

small: 40S, 34 proteins, 18S RNA

large: 60S, 50 proteins, 28S, 18S, and 5.8S RNAs
Where are proteins synthesized on?
On the large subunit of the ribosome. It involves complex ribonucleoprotein particles comprised of different subunits .
What is the P site, A site, and E site?
P site: peptidyl tRNA binding site for peptidyl-transfer

A site: acceptor site or aminoacyl-tRNA binding site

E site: exit site for growing peptide chain
What does the initiation phase of translation in eukaryotes require?
It require bringing together a small 40S ribosomal subunit, an mRNA, and a tRNA complex which must be in the correct orientation. The large subunit is associated with the complex to form a completed initiation complex. It requires initiation factors.
What are the steps of the initiation phase in translation?
1. eIF-2a binds to GTP and Met-tRNAi^met to form a ternary complex. The Met-tRNA can't be replaced by another tRNA

2. Ternary complex combines with a small ribosomal subunit which associated with eIF-3
*eIF-3 creates a binding location for the large subunit and blocks any other subunit association.

3. Interation with mRNA forms a pre-initiation complex. Additional IFs are associated:
- eIF-4f - cap binding complex
- eIF-4a - helicase
- PAB - polyA-binding protein
GTP is hydrolyzed by eIF-2a to form a complex which is released.

4. Large ribosomal subunit and eIF5b-GTP bind to pre-initiation complex
What are the steps of the elongation phase in translation?
1. Initiation complex (with Met-tRNA) in 80S P site.

2. GTP hydrolysis results in a conformational change in EF1alpha.

3. First peptide bond has been formed and occupies a hybrid A/P site on ribosome. The acceptor stem is in the E site.

4. mRNA-peptidyl rRNA complex is translocated to the P site. The deacylated initiator tRNA is moved to E site.

Elongation cycle continues until a STOP codon is reached!
When a termination codon in mRNA occupies the A site, what occurs?
eRF binds to GTP and forms a complex; deacylated tRNA is released.

Peptidyltransferase hydrolyzes ester bond and releases protein.

GTP is hydrolyzed to GDP and alters the conformation of the releasing causing the dissociation of the ribosomal subunits.
T/F: Proteins can't independently and spontaneously generate their correct 3D conformation.
FALSE. They can! Some proteins actually need the help of chaperones which reversibly bind hydrophobic regions of unfolded proteins and folding intermediates
Where are proteins destined for export synthesized on?
Membrane-bound ribosomes on the rough ER.
What are the components of secretory proteins?
- hydrophobic signal sequence near the amino terminus (charged N-terminal region with hydrophobic amino acids followed by a more polar C-terminal segment that acts as a cleavage site).
What is the function of a signal recognition particle (SRP)?
It's an elongated particle made up of 6 different proteins and a small RNA molecule.

It binds to the charged N-terminal region to stop protein synthesis.
Where are most mitochondrial proteins synthesized in?
cytosol.
What type of presequences mark the protein for the mitochondrion? Is it specific?
N-terminal presequences. It's not specific
What occurs in N-linked glycosylation?
1. Process begins on the cytoplasmic face of the ER membrane. Dolichol acts as the glycosyl acceptor of N-acetylglucosamine.

2. Transfer of N-acetylglucosamine to dolichol

3. 5 mannose sugars are added sequentially and the oligoscaccharide is oriented towards the lumeneal side of the membrane.

4. More mannose and glucose residues are added to complete the structure.

5. Structure is transferred to ASN residue on lumeneal membrane surface.
When does O-linked glycosylation occur?
It only occurs after the protein has reached the Golgi apparatus
T/F: Cleavage of precursor proteins is not common when activating enzymes and proteins.
FALSE. It IS common.
Can amino acids become modified after being incorporated into proteins? What are some of the processes involved if it can be incorporated?
Yes they can be modified reversibly or permanently.

Methylation, acetylation, reverse phosphorylation, ADP-ribosylation.
Where does O-linked glycosylation occurs?
It occurs only on fully folded proteins.

It's post-translational.
What is intercellular signal transduction? What are the types of intercellular signaling?
Intercellular transduction involves a involve being sent to other cells via the external environment.

Types: juxtacrine, paracrine, endocrine, synaptic, and autocrine
What is intracellular signal transduction?
Intracellular signal transduction involves the stepwise regulation of intracellular proteins that ultimately alter the function of the target cell.
What is juxtacrine signaling?
- contact-dependent

- direct transfer of small ions/metabolites btwn neighboring cells

- interaction is due to a protein on the surface of the sender cell and a receptor protein on the surface of the target cell
What is endocrine signaling?
- signaling between cells (short vs. long distance)

- high affinity of hormone-receptor interactions

- lonnnng time
What is paracrine signaling?
- secretes molecules into immediate environment

- rapid and localized communication between cells
What is synpatic/neuronal signaling?
- extreme type of paracrine

- shorter distance and restricted synaptic volume

- high concentration

- low affinity

- rapid termination
What is autocrine signaling?
- one cell type is both the sender and target cell

- used by embryonic/neontal organisms during development

- immune/inflammatory responses for adult organisms
What are some types of secreted signaling molecules?
proteins, glcoproteins, small peptides, aa, amines, lipids (FA & steroids), nucleotides, nucleosides, and gases
What kinds of molecules bind to intracellular receptor proteins?
- hydrophobic molecules (steroid hormones and vitamins)

- once bound, they enter the nucleus and regulate transcription
What kinds of molecules bind to cell surface receptors?
molecules that are too big (proteins and polypeptide hormones) or too hydrophyilic
What is a ligand? What is an agonist? What is an antagonist?
Ligand: any molecule that binds to receptor protein

Agonist: ligand that activates signal transduction when it binds

Antagonist: ligand that prevents signal trandsduction when it binds
What is a physiological agonist/antagonist? What is a pharmacological agonist/antagonist?
Physiological: ones that occur naturally as receptor ligands (hormones and NTs)

Pharmacological: synthetic molecules that act as receptor ligands (therapeutic drugs)
T/F: The same ligand can bind to different receptors yet have the same response.
FALSE! They have very distinct responses.
What are effectors?
Signaling proteins (enzymes/ion channels) that are distal to but are activated by the agonist-bound receptors

- receptor protein can be effector
What are second messengers? What are some examples?
2nd messenger: small intracellular molecules that transmit and amplify the initial signal from agonist-activated receptors

Examples: cAMP, cGMP, DAG, IP3)
Most pathways of intracellular transduction involve changes in ______ of certain proteins (think of protein kinase cascades!)
PHOSPHORYLATION!
What is the signaling mechanism for protein phosphorylation? How about the signaling for GTP-binding regulatory proteins?
Protein phosphorylation: input signal comes in and activates the kinase which hydrolyzes ATP to ADP. The Pi is bound and so the system is turned on --> output signal.
When phosphatase is activated, it removes the Pi and the system is now in its off position.

GTP-binding regulatory protein: Input signal comes in and GTP-binding activates so GDP is phosphorylated to GTP. GTP is bound to the system and the system is on --> output signal.
When GTP is hydrolyzed to GDP and the GDP is bound to the system, the system turns off.
What are some of the termination mechanisms for cell surface receptors?
Receptor inactivation:signal created but then goes back to surface receptor.

Receptor internalization: reduced # of cell-surface receptors because of engulfed by endosome

Receptor down-regulation: receptor is digested by lysosomes.
Explain what ligand-gated ion channel receptors do.
These receptors are found on the nerve-nerve or nerve-muscle synapses.

Very rapid signaling

Ligands bind to receptors with low affinity --> so you have rapid dissociation of ligand-receptor complex when there's a reduction of extracellular concentration.

Binding of NT triggers near-instantaneous change in receptor conformation that permits ions to move through the receptor protein complex
Enzyme-linked receptors
Intracellular domains have a catalytic domain (ex: protein kinases, protein phosphotases, proteases, or nucleotide phosphodiesterases)

Receptors regulate long-term cell functions (mins. - hrs) by initiating intracellular signalling cascades that culminate in activation of inhibition of gene expression
What is a process that a living cell must to do respond to changes in its environment?
Alter expression of specific genes.

*Important stuff:
- need to regulate developmental gene expression as well as tissue specific gene expression
Define following terms: operon, structural gene, regulatory gene, control elements.

Other terms: induction, repression. (These weren't in the notes but they were in the book)
1. operon - complete regulatory unit of a set of clustered genes.

2. structural genes - code for related enzyme or associated proteins

3. regulatory genes - code for regulator proteins

4. control elements - sites on the DNA that regulator proteins act on (near the structural genes)



induction: occurs when transcription of structural genes increases in response to specific substrate

repression: occurs when a specific protein prevents the transcription of certain structural genes
What are the control elements, structural and regulatory genes of the lactose operon?
control elements: CAP, lacP, and lacO
*lacP = promoter
*lacO = operator

structural: lacZ, lacY, and lacA

repressor: lacl
What do each of the structural genes in the lactose operon code for?

(This was in the book)
lacZ: Beta-galactosidase

lacY: permease (transport of sugars including lactose, across membrane)

lacA: codes for Beta-galactoside transacetylase (transfers an acetyl group from acetyl CoA to Beta-galactosides)


Side notes:
*galactosidase participates in the pathway
*transacetylase is associated with detox and excretion of nonmetabolized analogs of B-galactosides
In the lactose operon, what makes up lacP? What makes up lacO? Where is lacl located? Where is lacZ located?
lacP (promoter): CAP site + RNA polymerase interaction site

lacO: operator but it overlaps with lacP**

lacI is located upstream of the controlling elements

lacZ is located downstream of lacI
T/F: Transcription of lacI is regulated.
FALSE! It is not regulated, it's transcribed from its own promoter at a low rate that's independent of the cell's status

Side notes:
- strong affinity for lacO
- prevents RNA polymerase from binding to lacP and initiating transcription
- strong affinity for inducer (when inducer is present, it binds to repressor so repressor is kicked off of operator so transcription can begin)
Explain the mechanism of the lactose operon (when lactose IS present and when it is NOT present).
When lactose is present, lactose binds to the repressor causing a conformational change. Now, the RNA polymerase can transcribe the ZYGA genes --> RNA --> proteins!

When lactose is absent, the repressor binds to the operator and acts as a roadblock for RNA polymerase so it can't transcribe!
What is IPTG?
Isopropylthogalactoside -- serves as an inducer for the lactose operon but it's not metabolized by Beta-galatosidase
T/F: If you have a mutation of lacI, It will affect the affinity of the repressor for the operator.
True: some are changed (leads to continuous transcription) and while other changes don't affect the affinity.
In terms of the lactose operon, what is actually the natural inducer?
Allolactose!

Allolactose and lactose are composed of both galactose and glucose but the linkage is different.
*A side-rxn of Beta-galactosidase which breaks down lactose to galactose and glucose actually converts these two to allolactose.
What is immediately upstream of the operator in the lac operon?
The promoter which contains the CAP-binding site and the RNA polymerase binding site.
What happens to the lac operon when:
1. glucose and lactose are present
2. glucose is present and lactose is absent
3. glucose and lactose are absent
4. glucose is absent and lactose is present
1. since glucose is the preferred source of sugar, the operon is off since it inhibits AC -- CAP is not bound
2. operon is OFF because repressor is bound (since there's no lactose) and glucose inhibits AC so CAP is not bound
3. operon is OFF because there's no lactose to bind to the repressor (BUT since glucose is absent, AC is activated and generates cAMP to form a complex with CAP)
4. operon is ON. How? Glucose is absent so AC is activated and generates cAMP which binds to CAP and binds to the CAP binding site. Since lactose is present, it binds to the repressor and removes the repressor so the RNA polymerase can transcribe.
Lactose addition (decreases/increases) concentration of allolactose
increases
What actually binds to the repressor and induces the operon?
ALLOLACTOSE!! Not lactose.
T/F: E.coli prefers lactose over other sugars as a carbon source.

(In the book.)
FALSE! Glucose actually interferes with the induction of the lactose operon --> catabolite repression

*Glucose inhibits adenylate cyclase (so no synthesis of cAMP) --> lower intracellular concentration of cAMP
What does cAMP do in the lac operon?

(In the book.)
It forms a complex with CAP which binds the CAP regulatory site at promoters of lactose.

- Complex exerts a + control on transcription by causing the DNA helix to bend so the interaction between the complex and RNA polymerase can activate transcription.
A regulator ribosomal protein has a (low/high) affinity for the rRNA and (lower/higher) affinity for one or more regions of its own mRNA.
high, lower
T/F: If free rRNA is not available for assembly new ribosomal units, individual ribosomal proteins bind to monocistornic mRNA from their own operon, blocking further tranlsation
FALSE! They bind to polycistronic mRNA in bacteria
*Info on Gene Expression in Eukaryotes
some genes expressed in most cells
some genes are activated upon demand
some genes are permanently inactive in a few cell types
What were some important factors in the regulation of transcription in eukaryotes?
chromatin, DNA modification (methylation of DNA), and TFs (transcription factors)
What happens to genes that are not transcribed within a particular cell?
They form a highly condensed heterochromatin. If transcribed, they have a less condensed more open structure (active chromatin -- looser arrangements!)
What kind of residues do histones contain? And why is it important? What if you modify lysine?
They contain a lot of lysine residues (positively charged side groups) which interacts with the negatively charged phosphodiester linkages of DNA

If you modify lysine by acetylation, you make neutral and you weaken the electrostatic interaction. HOWEVER, acetylation is reversible. Acetylation and deacetylation just provides a way to loosen or tighten chromatin structure
What does methylation in human DNA center on?
Formation of 5-methylcytosine from cytosine in the sequence of CG on both strands.
About what percentage of CG sequences in human DNA are methylated?
70%
What does methylation of DNA often correlates with?
Inactivation of genes and lack of transcription because proteins recognize and bind methylated DNA which prevents binding of transcription factors.

Also, deacetylation of histones!
What kind of domains are in eukaryotic transcription factors?
1. DNA recognition - site-specifc binding
2. Activation - contact general TFs, RNA polymerase II, or other regulators
3. Dimerization - promote formation of homodimers/heterodimers with another monomeric TF
4.Protein interaction - association with other proteins
5. Domains that interact with coactivators
Explain the structure of HTH DNA.
One helix binds to the major groove of the double stranded DNA while the other helix binds to the sugar-PO4 backbone

- HTH DNA binding proteins recognize palindrome DNA sequeunces
What does a Zinc Finger motif contain?
Zinc atom bonded to 2 histidine and 2 cysteine side chains
What does TFIIA do? What does TFIID contain? What does TFIIE do? What does TFIIF do? What types of activities does TFIIH perform?
It's a complex that contains TBP and different TAFs
When is the LDL receptor gene transcribed?
When there is a lack of cellular cholesterol
What does increased transcription of the LDL receptor gene lead to?
increased amount of LDL receptor and enhanced uptake of LDLs and their cholesterol
What makes up the Sp1 protein? What is recruited to activate Sp1?
zinc finger motif and glutamine-rich activation domain to help assist in the recruitment of TFIID to TATA.

CRSP (cofactor recruised Sp1)
What does activation of the LDL receptor gene need?
- binding of Sp1
- SREBP-1a binds to sterol response element sequence
T/F: SREBP is synthesized as a precursor protein that doesn't need to be protelytically processed before it acts as a TF.
FALSE! It must be proteolytically cleaved!
What is SREBP in close association with (what other protein)?
SCAP - SREBP cleavage activating protein
- remains unprocessed when cholesterol levels are normal
- SCAP senses low levels of cholesterol and moves with SREBP to cis-Golgi where two proteases cleave SREBP
What is recruited to the promoter region once SREBP, Sp1, and CRSP bind?
The histone acetyltransferase, CBP
What is the purpose of the polymerase chain reaction?
Spit out large quantities of specific DNA sequences
What does the PCR require?
Two nucleotide oligomers that serve as primers for DNA polymerase to extend
What is the mechanism of the PCR?
- Unknown sequence of DNA fragment is inserted into a plasmid vector of known sequence
- DNA is heated to 90 deg C to dissociate the double strands and then cooled so two complementary oligonucleotides to each strand are inserted so it can hybridize to the known DNA sequences
- dNTP's and DNA polymerase are added
- steps are repeated to yield more copies
What kind of DNA species serve as templates for DNA replication in PCR? What is the product of PCR?
single-stranded!

a double-stranded DNA molecule
What type of heat-stable polymerase is used to help amplify the PCR with each DNA molecule?
Thermal stable polymerase: Taq DNA polymerase
What temperature is the DNA denatured at? What temperature do the strands anneal at? What is the extension temperature?
90, 55, and 72 deg C
Explain what recombinant DNA is.
It allows for the removal of a piece of DNA out of a larger complex molecule and amplification of the DNA fragment.

- Restriction endonucleases hydrolyze DNA in a staggered way to produce "sticky" ends to yield single-stranded fragments.
- Ends can hybridize or can anneal together
- DNA ligase joins the two fragments to produce a recombinant DNA molecule
What is the whole point of directional cloning?
To reduce the number of variable "recombinants" and increases the probability of selecting the desired recombinant.
Explain the process of directional cloning.
Insert foreign DNA into a plasmid vector without the plasmid resealing itself via two restriction endonucleases.
- these two enzymes yield DNA fragments and linearized plasmids with different sticky ends (plasmid can't reanneal with itself)
- now the foreign DNA is inserted into the vector in one orientation
Explain the Sanger Procedure of DNA sequencing.
Based on the random termination of a DNA chain during enzymatic synthesis

- DNA region of interest is inserted into bacteriophage DNA molecule
- the region that flanks the DNA of interest contains a sequence complementary to universal primer
- the replicating bacteriophage produces a ss recombinant DNA molecule readily purified (the known sequence downstream of the DNA insert serves as a hybridization site)
- extension of the primer is catalyzed with a DNA polymerase and all four dNTPs + 1 ddNTP
- synthesis stops whenever a ddNTP is incorporated into the growing molecule

**Sanger procedure and PCR methods can be combined for direct sequencing of the DNA regions of interest.
Why are transgenic animals used?
transgenic animals are animals that develop from such a fertilized egg that carry the inserted gene in every cell


To help investigate the role of a selected gene in growth and development of a whole animal
- gene is introduced in the fertilized egg
Explain the method to create transgenic animals.
- gene of interest is a cloned recombinant DNA molecule with its own promoter
- multiple copies are microinjected into fertilized egg
- foreign DNA inserts randomly within chromosomal DNA
- implant eggs into foster mother mouse
What is the purpose of knockout mice?
To help identify the biological role of a gene by knocking out a certain gene
Explain the method of creating knockout mice.
- cultured embryonic cells are manipulated via recombinant technology carry a defective gene
- altered cells are introduced into a blastocyst which is implanted into a foster mother
What is the purpose of nick translation? Explain the method of use.
To label DNA probes to help detect the presence of complementary RNA or DNA in experimental samples

1. nicking step: introduces random ss breaks in DNA
2. translation step: E.coli DNA polymerase I has both 5'-3' exonuclease activity to remove nucleotides from 5' end of the nick and polymerase activity that simultaneously fills in the gap with radioactive nucleotides using the 3'-end as a primer.
What is the purpose of the Southern blot technique?
Transfer DNA to nitrocellulose as ss molecules to detect specific DNA sequences within a complex mixture.
- hybridization with nick-translated labeled probes can show if a DNA sequence of interest is present in the same or different regions of the genome
What is the purpose of EMSA? Explain the method of use.
To analyze sequence-specific DNA-binding proteins along with the DNA sequence required for binding

- proteins with DNA-binding characteristics are prepared
- DNA probe is radiolabeled
- DNA employed may be DNA fragments, etc. (must be double-stranded)
- the purified labeled DNA and protein sample are preincubated to form a stable pr-DNA complex
- Purified DNA migrates through gel when there's an electric field
- then if pr is added that forms a complex with this DNA, the MW of the DNA is increased
- this would slow its migration through the gel (mobility shift)
- so if you add an antibody to this complex, it will increase the size and slow its migration
- if antibody reacts with the DNA-binding region of the protein, a DNA-pr complex won't form
What is the purpose of NPA (nuclease protection assay)?
Analysis of multiple mRNA species

- ssDNA probes that are complementary to the known sequences of the different RNA transcripts are hybridized
- ribonuclease will remove the ssRNA regions of mRNA that didn't hybridize with the DNA probe
- DNA-RNA hybrids protected against the nuclease will remain for analysis by the acrylamide gel electrophoresis