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What are the three steps of transcription?

Initiation, elongation, and termination

1

Enzyme for DNa replication

DNA polymerase

2

How does the DNA polymerase add a nucleotide? (Chemistry)

The 3' oh attacks the alpha phosphate of an incoming dNTP

3

Role of the exonuclease

It has a high affinity for incorrect pairs and degrades the DNA from the 3' end

4

Processive versus non-processive

//

5

When does chromosome replication occur?

In the s phase of the cell cycle

6

End replication problem

Since DNA synthesis needs an RNA primer to initiate DNA strands, the ends of DNA will have a hard time replicating.

7

Telomeres

In eukaryotes, to fix the end replication problem, they have telomeres which are TG rich seq that doesn't code for anything

8

Telomerase

A special DNA polymerase that only creates the telomeres. It has an RNA component and so it is a ribonucleoprotein and doesn't need an template to add bases. It's also like a reverse transcriptase because it uses the RNA template to make dna

9

What end does the telomerase act on?

It acts on the 3' end

10

General causes of mutation

Environmental factors like chemicals and UV light, tautomerization, wrong base pairing, transposons, inaccuracy in replication

11

Transition mutation

When a purine is switched with another purine or pyrimidine with pyrimidine

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Transversion mutation

Purine to pyrimidine switch

13

Point mutation

Mutations that alter a single nucleotide

14

Why are DNA micro satellites prone to mutation?

DNA microsatellites are long repeating seq like CACA and they are harder to replicate because slippage may occur. Therefore the repeat length may vary depending on the individual

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Mutagen

A chemical that increases the rate of mutation

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Deamination of cytosine

This is one of the most common mutations and turns cytosine into uracil

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DNA depurination

This is when there is spontaneous hydrolysis of the N glycosyl linkage and it produces a deoxyribose without the base

18

Thymine dimer

When two thymine bases fuse to make a cyclobutane ring and this causes DNA polymerase to stop during replication if it reaches this point

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How do gamma radiation and x-rays damage DNA?

They break the double strand which is really difficult for the cell to repair

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Intercalating agents

They are able to slip between bases and cause errors in replication. They do this by causing additions, deletions, or even frame shifts

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Base excision repair

DNA glycosylase removes the base first, and then AP endo and exo nuclease remove the backbone. The gap is then filled with the correct base by DNA polymerase.

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Photoreactiviation

Reverses the formation of pyrimidine dimers from uv radiation by using the energy from light directly to break the dimer bond

23

Nucleotide excision repair

The DNA is scanned by a tetramer (UvrAB) for distortions and if a distortion is detected, UvrC will cleave a segment with the lesion out.

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Methyltransferase

It reverses the methylation of guanine by taking the methyl group and putting on its own cysteine

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What can mutations in the genes coding for nucleotide excision repair cause?

Can lead to UV light sensitivity

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Double strand break repair

Usually repaired by non homologous end joining but during replication is is fixed by homologous recombination.

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Non-homologous end joining

Ku70 and Ku80 bind to the broken ends and recruit DNA PKcs which then recruit Artemis, an exo and endonuclease that will process the broken ends. Then lipase is recruited to to seal the ends together.

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Homologous recombination

The broken strand uses the original parent strand as a template and then the copied area is switched with the template so now the repaired strand has part of the template strand within it.

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DNA cloning

Selective amplification of a particular gene or DNA segment

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The five general steps of DNA cloning

1) cut DNA at specific location (restriction endonuclease)
2) select a cloning vector that can self replicate
3) join your DNA fragment with the vector
4) put this recombinant DNA into a host organism
5) select the cells that have the recombinant DNA

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What do you need for a successful DNA vector?

You need an origin of replication, a selectable marker, and a region in which your DNA can be inserted

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Plasmid

A circular double stranded DNA molecule with a size from 1 to 200 kb. They replicate separately from the host cells chromosomal DNA

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How to insert your DNA into plasmid

Use same cleaving enzyme like EcoR1 to make sticky ends. Then allow your DNA fragments to anneal and use ligase to seal them together

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How to get vector into host cell

Use transformation for bacterial cells and transfection in mammalian cells

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BACs and YACs

They are used to clone large segments. If you clone these within the lacZ gene, you can tell if the insert is present or not.

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How to tell if BAC or YAC has insert

If it does have the insert, the lacZ gene will be disrupted and won't turn color in media with x-gal.

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DNA library

1) DNA digested with restriction enzyme
2) cleaved DNA mixed with vector ligase
3) this creates a DNA library because each vector has different DNA fragment. You can then screen for your gene of interest

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Genomic versus cDNA library

A genomic library represents the entire genome while a cDNA library represents only the genes expressed in the cell since the mRNA was copied into DNA

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Reverse transcriptase

Converts RNA sequences into DNA. Needs a primer and either DNA or RNA template . You can make tissue specific cDNA libraries

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RNA polymerase in prokaryotes

Has beta core

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RNA polymerase in eukaryotes

There's I, II and III but we focus mostly on RNAP II because it is involved in mRNA synthesis. The others make RNA for ribosomes and tRNA

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How many metal ions are involved in RNA polymerase?

Two Mg2+ ions are involved but one comes with the incoming rNTP

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What direction is RNA made in?

From 5' to 3'

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Template strand versus the coding strand

The template strand is the one the RNA will be made complementary to, while the coding strand is the one the RNA is identical to.

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Antisense versus the sense strand

Antisense = template strand
Sense = coding strand

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What does upstream mean?

It means toward the 5' end of a given sequence

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RNA polymerase holoenzyme

Only in prokaryotes, consists of the RNA polymerase core (5 subunits) and the sigma factor

48

Job of sigma factor

It recognizes the promoter region and helps the RNA polymerase bind to the promoter region.

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Promoter region (types)

The most common promoter region has a -10 and -35 element. A stronger promoter might have a UP element. The third type has no -35 but instead has an extended -10. The last type has a discriminator next to the -10 region.

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What recognizes the UP element?

Alpha CTD (or the carboxyl terminal domain of the alpha subunit)

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Steps within transcription initiation

Closed complex = binding of sigma factors to promoter region
Open complex = DNA begins to be unwound
Initial transcript = short RNAS are made and released repeatedly until elongation phase begins

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How does the RNA polymerase transition to the open complex?

The sigma 1 factor moves out of the active site and the promoter melts

53

Steps of transcriptional elongation

RNA polymerase unwinds the DNA at the front, adds rNTPs, reanneals the DNA in the back, proofreads and dissociates the mRNA from the template

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Hydrolytic editing*

The nuclease within the RNA polymerase cleaves off an entire segment of ribonucelotides

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Pyrophosphorolytic editing

Look in book

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Terminator

A sequence that triggers the RNA polymerase to dissociate from DNA and release the RNA

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Rho independent terminator

A sequence rich in GC that will form a hairpin and cause a release of the polymerase without the need of a protein

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Rho dependent terminator

When the protein factor rho is present, RNA synthesis will stop and every dissociates. These sequences have a rut site that rho binds to.

59

GTF'S

They are general transcription factors that are needed in eukaryotes to recognize and bind the promoter. Somewhat similar to the sigma factor in prokaryotes

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The transcription factors needed for pol II

TFIID, TFIIA, TFIIB, TFIIF, TFIIE, TFIIH

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The main promoter for eukaryotes

TATA box

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What binds to the TATA box first?

TBP and TFIID bind first. They then recruit other GTF's and pol II which forms the ore initiation complex

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Steps for initiation in eukaryotes

Closed complex = melting of the promoter by TFIIH (needs ATP)
Open complex = phosphorylation of the polymerase tail by TFIIH which also needs ATP
Promoter escape = release from promoter and most general transcription factors

64

What is the last step needed before elongation occurs?

The mediator complex connects the activator sequence to the initiation complex. Chromatin remodeler and HAT are also needed

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What happens in eukaryotic elongation?

The transcription initiation factors and mediator complex are shed and new proteins are recruited. Different phosphorylation of the tail exchanges initiation factors for the new proteins

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What phosphorylation signals the capping enzyme in the promoter escape phase?

Phosphorylation of serine 5

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What phosphorylation signals the splicing machinery?

Serine 2 phosphorylation

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5' cap

A modified guanine is added to the 5' end of the mRNA. The beta phosphate of the chain attacks the alpha phosphate and creates a 5' to 5' linkage. This guanine is then methylated (modified). All eukaryotic mRNAs have a cap

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Polyadenylation

Poly A tail is added to the 3' end. CstF and CPSF bind to the tail, and then to the RNA when signal sequence is transcribed. CstF leaves after it cleaves the RNA and PAP is recruited. PAP adds about 200 Adenines to the RNA without a template and finally polyA binding proteins bind to the tail

70

Torpedo model

After the first RNA strand is cleaved off to make the polyA tail, the uncapped RNA is degraded by RNase and this destabilizes the RNA polymerase and causes termination.

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Allosteric model

Once the polyA signal is passed, the RNA polymerase becomes less processive because of a conformational change

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Function of polyA tail and 5' cap

They stabilizes the mRNA, allow it to be transported out of the nucleus, and help with translation initiation

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Gene expression is prokaryotes versus eukaryotes

In prokaryotes, transcription and translation can occur simultaneously. While in eukaryotes, there is transcription, then RNA processing, then transport, and finally translation.

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Exon

Sequences that contain the genetic information and remain present within the final RNA product

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Introns

Intervening sequences that are removed by RNA splicing

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hnRNA

Heteronuclear RNA. RNA of many different sizes

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Pulse chase experiment of RNA

In the pulse, RNA was labeled with 32P for 30 minutes. The RNA was then separated by rate zonal centrifugation. In the chase, they stopped transcription and allowed the cells to grow for 3 hours. They concluded that mRNA is derived from hnRNA.

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Signals for RNA splicing

There's the 5' splice site, 3' splice site and the branch site. They occur at exon/intron boundaries in most genes

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The splicing reactions

It includes two transesterification reactions.
1) 5' splice site is cleaved
2) the branching point OH attacks the 5' end phosphate and forms a loop (lariat) 5' to 2' bond
3) the 3' end is cleaved
4) the 5' exon attacks the 3' exon and they join together

80

What is the spliceosome made of?

Made of SnRNPs (small nuclear ribonucleoprotein particles) U1,2,4,5,6 which catalyze the reactions. They are ribozymes!

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Which SnRNP recognizes the 5' splicing site?

Usually U1

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The steps of the spliceosome mediated splicing reaction

E complex, a complex, b complex, and finally the c complex

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What happens in the E complex of splicing reactions?

U1 recognizes the 5' binding site, BBP binds to the branching point, and U2AF binds to the py tract and the 3' splice site

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What happens in the A complex of splicing?

U2 comes in and replaces BBP

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B complex of the splicing reactions

U4, U5 and U6 bind to the intron and form a complex with the others. U1 leaves and U6 takes its place at the 5' splice site. U4 is then released and this allows U6 to interact with U2

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C complex of RNA splicing

U6 and U2 catalyze the first transesterification reaction and then U5 helps with the second one.

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A rare form of RNA splicing

Some rare RNA have the ability to splice themselves. They can do this by folding into a conformation that catalyzes it's own release from the surrounding exons

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Alternative RNA splicing

Occurs in 95% of human genes with introns. Allows multiple proteins to be made from one gene. It is often cell type specific.

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Introns early model

Originally prokaryotes had introns too but lost them over time in order to replicate more efficiently.

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Introns late model

Introns were inserted into genes that originally did not have introns

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Exon shuffling

Allows the creation if new genes through duplication and recombination. It is one reason why introns have been kept in our genome.

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What do you need for translation? (Key components)

mRNAs, tRNAs, and the ribosome

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Three stages of translation

Initiation, elongation, and termination. Same as transcription!

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How many codons are there?

There are 61 codons coding for 20 amino acids plus 3 stop codons for a total of 64 codons.

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How many possible reading frames does mRNA have?

3 possible reading frames

96

Open reading frame

Starts with a start codon and ends with a stop codon

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Which direction are codons read?

5' to 3'

98

Silent mutation

When a nucleotide is switched but it still codes for the same amino acid

99

Missense mutation

When a nucleotide is switched and codes for a completely different amino acid

100

Nonsense mutation

When a nucleotide is switched and the new codon is now a stop codon

101

Which end of the tRNA holds the amino acid?

The 3' end. It usually ends with CCA

102

Uncharged versus charged tRNA

Uncharged doesn't have an amino acid and charged does

103

Secondary and tertiary structure of a tRNA

Secondary is like a cloverleaf with anticodon loop, variable loop, pseudouridine loop, d loop and acceptor arm. The tertiary structure looks like an L with the acceptor arm on one end and the anticodon loop on the other end

104

What enzyme charges the tRNAs and how does it do it?

Aminoacyl-tRNA synthetase requires ATP in order to add the amino acid to the tRNA. (20 different synthetases)

105

Adenylation

The carboxylic acid of the amino acid attacks the alpha phosphate of the adenine to make an adenylylated amino acid. This process requires ATP

106

How does tRNA charging occur? (Chemical process)

The adenylylated amino acid is attacked by the 3' OH of the tRNA. This process releases the AMP.

107

Size of ribosome subunits in prokaryotes

The large subunit is 50S and the small subunit is 30S. This adds to 70S

108

Ribosome subunit size in eukaryotes

The large subunit is 60S and the small subunit is 40S to make a total of 80S

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The ribosome cycle

Initiation, elongation, and termination

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Polysomes

When multiple ribosomes can translate a single mRNA molecule simultaneously

111

Function of the small ribosome subunit

It mediates the interaction between the codons and tRNA anticodons

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Function of the large ribosome subunit

Catalyzes the peptide bond formation, and has binding sites for G proteins that assist in initiation, elongation, and termination

113

Peptidyl transferase reaction

The reaction that adds the next amino acid to the chain (creating a peptide bond)

114

What direction are polypeptides synthesized?

From the n terminus to the c terminus

115

The structure of a ribosome

Has an a site or aminoacyl site, the p site (peptidyl) and finally the e site (exit)

116

Prokaryotic translation initiation

The 16s rRNA be pairs with the ribosome binding site (RBS) and this positions the start codon with the p site of the ribosome

117

Polycistronic

When mRNA contains multiple open reading frames

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Initiation in prokaryotic translation

The small subunit binds to the start codon and then the initiator tRNA binds to the AUG and has a special methionine. The large subunit then comes in to form the complete ribosome

119

Function of IF3, IF2, and IF1

IF3 binds the e site and doesn't allow the 50S to bind. IF1 blocks aminoacyl tRNA from binding to the a site. IF2 is a gtpase that interacts with IF1 and the special methionine tRNA. This leaves only the p site open for the initiator tRNA

120

Monocistronic

MRNAs with only one open reading frame

121

Why are eukaryotic mRNAs held in circles? And how?

This is so the ribosome can easily reassociate to make more protein. It is held in a circle by the binding of elF4A to the poly A tail

122

Release factors for translation in prokaryotes

The class 1 RFs release the peptide chain from the last tRNA. Class 2 RFs release the class 1 factors.

123

RRF

Ribosome recycling factor helps release the tRNAs and dissociate the ribosome subunits