Exam 2 Memory Dump - Lecture Slides

What are the steps of molecular/DNA cloning?

1.) Cloning Vector is cleaved with restriction endonuclease

2.) DNA fragment of interest is obtained by cleaving chromosome with a restriction endonuclease.
3.) Fragments are ligated to the prepared cloning vector via DNA ligase

4.) DNA is introduced into the host cell through a recombinant vector
5.) Propagation (cloning) of transformed cell produces many copies of recombinant DNA.

What happens when DNA molecules are cleaved by restriction restriction endonucleases?

When Type II restriction endonucleases cleave DNA, they leave either sticky ends (with protruding single strands) or blunt ends. The restriction fragments can be ligated to other DNAs, such as the plasmid cloning vector shown here. Ligation is facilitated by the annealing of complementary sticky ends, and it is less efficient for DNA fragments with blunt ends than for those with complementary sticky ends. DNA fragments with noncomplementary sticky ends (i.e., those created by different restriction enzymes) generally are not ligated.

What are DNA polylinkers and what can be done with them?

A synthetic DNA fragment with recognition sequences for several restriction endonucleases—a fragment known as a polylinker—can be inserted into a plasmid that has been cleaved by a restriction endonuclease.

What are important features of the first constructed E. coli plasmid pBR322?

1. The plasmid pBR322 has an origin of replication, or ori: a sequence where replication is initiated by cellular enzymes (see Chapter 11). This sequence is required to propagate the plasmid. An associated regulatory system is present that limits replication to maintain pBR322 at a level of 10 to 20 copies per cell.

2. The plasmid contains genes that confer resistance to the antibiotics tetracycline (TetR) and amplicillin (AmpR) allowing the selection of cells that contain the intact plasmid or a recombinant version of the plasmid

3. Several unique recognition sequences in pBR322 are targets for restriction endonucleases (PstI, EcoRI, BamHI, SalI, and PvuII), providing sites where the plasmid can be cut to insert foreign DNA.

4. The small size of the plasmid (4,361 bp) facilitates both its entry into cells and the biochemical manipulation of the DNA. This small size is generated simply by trimming away many DNA segments from a larger, parent plasmid—sequences that the molecular biologist does not need.

What are the steps of cloning DNA using selection with pBR322?

1. pBR322 is cleaved at the AmpR element by Pstl.

2. DNA fragments to be cloned are ligated to cleaved pBR322. Where ligation is successful, the AmpR element is disrupted. The TetR element remains intact.

3. E. Coli cells are transformed, then grown on agar plate containing tetracycline to select for those that have taken plasmid.

4. Individual colonies are transferred to matching positions on additional plates. One plate contains tetracycline, the other tetracycline and ampicillin.

5. Cells that grow on tetracycline (control), but not on tetracycline + ampicillin (negative selection) contains recombinant plasmids with disrupted ampicillin resistance, hence the foreign DNA. Cells with pBR322 without foreign DNA retain ampicillin resistance and grow on both plates.

What are the steps of building a cDNA library from mRNA?

1.) mRNA Template is annealed to synthetic oligonucleotide (oligo-dT) primer
2.) Reverse transcriptase and dNTPs yield a complementary DNA strand

3.) mRNA - (DNA hybrid) is degraded with alkali
4.) To prime synthesis of a second strand, an oligonucleotide of known sequence is often ligated to the 3’ end of the cDNA
5.) DNA polymerase I and dNTPs extend the primer to yield double-stranded DNA

What are the steps of PCR specific amplification and its ingredients?

Amplification

1. Heat to separate strands

2. Add Synthetic oligonucleotide primers; cool.

3. Add thermostable DNA polymerase (Taq) to catalyze 5’ → 3’ DNA synthesis

Repeats steps 1 and 2

DNA Synthesis (Step 3) is catalyzed by the thermostable DNA (Taq) polymerase (still present).

Repeat steps 1 through 3

After 25 cycles, the target sequence has been amplified about 10^6-fold

Insertion

1. Heat to separate strands

2. Anneal primers containing noncomplementary regions with cleavage site for restriction endonuclease

Clone by insertion at an EcoRI site in a cloning vector

PCR ingredients usually include: template DNA, primers, DNA polymerase, nucleotides, buffer, and Mg²⁺.

What are the steps of RT-PCR and its ingredients?

1. Isolate RNA – Extract RNA from the sample.

2. Reverse transcription – Use reverse transcriptase to convert RNA into cDNA.

3. Set up PCR – Add cDNA, primers, DNA polymerase, nucleotides, buffer, and Mg²⁺.

4. Denaturation – Heat to separate DNA strands.

5. Annealing – Cool so primers bind to the target sequence.

6. Extension – Polymerase copies the DNA.

7. Repeat cycles – Repeat denaturation, annealing, and extension to amplify the target.

8. Analyze product – Check the amplified DNA, often by gel electrophoresis or fluorescence.

RNA → cDNA → PCR amplify.
RT-PCR ingredients usually include: RNA template, Reverse transcriptase, Primers, dNTPs, DNA polymerase, Buffer, Mg²⁺, RNase-free water

What is the purpose of PCR?

PCR amplifies a target DNA segment so it can be detected and analyzed, basically making many copies of a specific DNA sequence,

What are the temperatures involved in PCR?

1. Denature DNA @ 95 C

2. Annealing temperature = Tm C – 5 to 10 C (50–65°C)

3. Add Taq polymerase (synthesized DNA) = 72 C

What is the purpose of RT-PCR?

To detect and amplify RNA by first converting it into cDNA.
Also:
Measure or detect gene expression

Detect RNA viruses

Study mRNA transcripts

Tells you what mRNA you isolated/collected and can evaluate it, as well as the amount of mRNA

What are the temperatures involved in RT-PCR?

Reverse transcription: about 42–55°C - Reverse transcriptase converts RNA into cDNA.
Denature DNA @ 95 C

Annealing temperature = Tm C – 5 to 10 C (50–65°C)

Add Taq polymerase (synthesized DNA) = 72 C

~42–55°C → 95°C → 50–65°C → 72°C

What are overall steps involved in Sanger DNA Sequencing?

1. Prepare template DNA – Use the DNA to be sequenced.

2. Add primer – A short primer binds to the template.

3. Add DNA polymerase, normal dNTPs, and fluorescent or labeled ddNTPs.

4. DNA synthesis begins – Polymerase extends the primer.

5. Chain termination occurs – When a ddNTP is added, extension stops.

6. Generate fragments of different lengths – Each ends at a specific nucleotide.

7. Separate fragments by capillary electrophoresis.

8. Detect the labeled fragments to determine the sequence.

9. Read the sequence from smallest fragment to largest.

Template + primer + polymerase + dNTPs + ddNTPs → extension → random termination → fragment separation → fluorescence detection → sequence readout

What is the purpose of Sanger DNA sequencing?

The purpose of Sanger DNA sequencing is to determine the exact nucleotide order of a DNA fragment.
Scientists use it to:

identify unknown DNA sequences

confirm cloned DNA or PCR products

detect mutations or sequence changes

verify whether a gene or DNA fragment is correct

Define and describe the purpose of the ddNTP, how is it chemically different that dNTP and how it is used in sequencing?

A ddNTP is a dideoxynucleotide triphosphate, a modified nucleotide used in Sanger DNA sequencing to stop DNA strand elongation.

• ddNTP = chain-terminating nucleotide

• Difference from dNTP = no 3′ OH

• Use in sequencing = stops DNA synthesis at specific bases so the sequence can be read

What are the general steps involved in DNA replication at both the leading and lagging strands?

1. Origin of replication opens

2. Helicase unwinds the DNA double helix

3. Single-strand binding proteins stabilize the open strands

4. Topoisomerase relieves supercoiling ahead of the fork

5. Primase lays down RNA primers

6. Leading strand synthesis begins

DNA polymerase synthesizes the leading strand continuously in the 5′ to 3′ direction, moving toward the replication fork.

7. Lagging strand synthesis begins

Because DNA polymerase can only synthesize 5′ to 3′, the lagging strand is made discontinuously, away from the fork, in short segments called Okazaki fragments.

8. More primers are added on the lagging strand

9. DNA polymerase extends each Okazaki fragment

10. RNA primers are removed

11. DNA ligase seals the backbone.

12. Proofreading occurs

13. Replication ends

What is the origin of replication (ori)?

the specific DNA sequence where DNA replication begins. Proteins bind here first to open the DNA.

What is the replication bubble?

the opened-up region of DNA formed after replication starts at the ori. The two strands separate in this area, creating a bubble-shaped region.

What is the replication fork?

the Y-shaped structure at each end of the replication bubble where the DNA strands are being unwound and new DNA is being synthesized.

Because DNA is a double helix, what must happen?

replication must synthesize two strands simultaneously in both the 5′→3′ and 3′→5′ directions.

What is similar and different between the directional building blocks of DNA strands, Nucleoside and Nucleotide?

Nucleotide = Sugar + Base
Nucleoside = Sugar + Base + Phosphate

Describe the DNA polymerase reaction.

a) DNA polymerases require a primer strand and a template strand (i.e., a primed template). As dNTPs are added to the 3′-OH group of the primer strand, this strand grows in the 5′→3′ direction. Incoming dNTPs are complementary to the template strand. (b) The insertion and postinsertion sites in DNA polymerase properly align the primed template for sequential addition of incoming nucleotides.

What does DNA Helicase do?

Unwinds the helix and pulls apart the double strands so primase, polymerase and ligase have access to single strands at one time

Helicases moves directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands (i.e. DNA, RNA, or RNA-DNA hybrid) using energy derived from ATP hydrolysis.

What does Topoisomerase do?

Catalyzes the removal of supercoils created by helicase.

Topoisomerases are important both in growing fork movement and in resolving (untangling) finished chromosomes after DNA duplication.

Type I topoisomerases relax DNA (i.e., remove supercoils) by nicking and closing one strand of duplex DNA.

Type II topoisomerases change DNA topology by breaking and rejoining double-stranded DNA.

What does DNA Primase do?

Lays down the initial RNA primers so that Polymerase III can get to work, and begin replicating.

Primase is an RNAP enzyme involved in the replication of DNA.

Primase catalyzes the synthesis of a short RNA segment (called a primer) complementary to a ssDNA template.

What happens to RNA primer in the later stages of DNA replication?

It is later removed by RNase H.
Once the primer is no longer needed it is removed.
RNAse H enzyme recognizes & removes the RNA primer.

Recognizes RNA:DNA hybrids

All except the last rNTP is removed

The last is removed by DNA polymerase exonuclease activity

Once removal is complete the sequence is filled in by DNA pol and the nick from the original removal is fixed to DNA ligase

What does DNA Ligase do?

Glues together pieces of DNA that have been created by Polymerase I and III.

It adds phosphate in the remaining gaps of the sugar-phosphate backbone.

What does SSB (Single Stranded Binding Protein) do?

binds to the separated single DNA strands during replication, stabilizes them, keeps them from re-annealing, and protects them from degradation.

What does DNA Polymerase do?

Catalyzes the polymerization of deoxyribonucleotides into a DNA strand.

DNA polymerases use a magnesium ion for catalytic activity.

Adds free nucleotides only to the 3' end of the newly forming strand.

This results in elongation of the newly forming strand in a 5'-3' direction.

No known DNA polymerase is able to begin a new chain (de novo).

DNA polymerase can add a nucleotide only onto a pre-existing 3'-OH group, and, therefore, needs a primer at which it can add the first nucleotide.

What is a Clamp Loader and what does it do?

It is is a protein complex in DNA replication that uses ATP to open and place the sliding clamp onto DNA at a primer-template junction. It then holds DNA polymerase on the DNA so the polymerase can copy DNA more processively without falling off

What are the two important function that DNA polymerase performs?

1. Deoxynucleotide polymerization

2. Editing/proof-reading

Describe why the conformational change in DNA polymerase is needed and how it serves help its function.

Only a correct conformation will allow for catalysis

Correct fits allow for a rapid enzymatic reaction & dNTP incorporation

Explain what the “fingers” do in regards to the “hand” metaphor representation of DNA polymerases I

Bind the incoming dNTPs

Once bound: they close to promote the catalysis of the incorporation

Interacts and guides the template strand

Explain what the “thumb” do in regards to the “hand” metaphor representation of DNA polymerases I

Interacts with the newly synthesized DNA

Maintains position of the primer and the active site

Maintains a strong association between the DNA & the active site when the fingers are opening to allow an additional dNTP to come in to the polymerase

Explain what the “palm” do in regards to the “hand” metaphor representation of DNA polymerases I

Contains the primary elements for the catalytic events

It binds to 2 divalent cations (Mg2+ or Zn2+)

One ion decreases the affinity of the 3 ́OH for its hydrogen

The other stabilizes the – charge of the B (beta) & y (gamma) phosphates that are released

Describe the role of Mg2+ and Asp in the polymerases

Mg²⁺ and Asp residues help DNA polymerase catalyze nucleotide addition using the two-metal-ion mechanism.

Asp residues in the active site bind and position the Mg²⁺ ions.

One Mg²⁺ helps deprotonate/activate the 3′ OH of the growing strand so it can attack.

The other Mg²⁺ helps stabilize the negative charges on the incoming dNTP phosphates and the leaving group.

Describe how each nucleotide is added

1. The correct dNTP base-pairs with the template base.

2. The 3′ OH of the growing DNA strand attacks the α-phosphate of the incoming dNTP.

3. A phosphodiester bond forms.

4. Pyrophosphate (PPi) is released.

5. The strand is now one nucleotide longer, always in the 5′ → 3′ direction.

Explain what occurs in base pair mismatches and how that compares/contrasts to correctly paired bases.

With correctly paired bases, the incoming nucleotide forms proper Watson–Crick hydrogen bonds with the template base, fits the polymerase active site well, and is efficiently added to the growing strand.

With a base-pair mismatch, the bases do not pair correctly, so the geometry is distorted, the fit in the active site is poorer, and polymerase is more likely to slow down, reject the nucleotide, or trigger proofreading to remove the wrong base.

What does DNA Polymerase Proof-reading provides in DNA replication?

Enzyme fidelity

What happens after nucleotide binding?

DNA polymerase undergoes a conformational change (fingers tighten around the active site)

What does an incorporation of an incorrect nucleotide means?

The positioning of the 3 ́-OH is incorrect

DNA synthesis cannot proceed (DNA polymerase requires a perfectly base-paired 3 ́ end as its substrate)

What are the two catalytic sites on DNA polymerases?

Polymerase active site – adds nucleotides to the growing DNA strand

3′→5′ exonuclease active site – removes mispaired nucleotides during proofreading

Explain the steps in the 3′→5′ proofreading exonuclease of DNA polymerases.

1. Polymerase mispairs dC with dT

2. Polymerase repositions the mispaired 3’ terminus into the 3’ → 5’ exonuclease site

3. Exonuclease hydrolyzes the mispaired dC.

4. The 3’ Terminus repositions back to the polymerase site

5. Polymerase incorporates the correct nucleotide, dA

What are Telomeres and what do they do?

Do not contain genes

Consist of multiple repeats of one short nucleotide sequence

Example is TTAGGG for humans

Number of repetitions is 100-1000+

Telomeres protect genes from being eroded by successive rounds of replication.

Telomeres also prevent cell from recognizing the ends as damaged.

What is Telomerase and what do they do?

Telomerase is a special enzyme that catalyzes the lengthening of telomeres

Telomerase consists of protein plus RNA

RNA contains a sequence that acts as the template for new telomere segment

Telomerase not present in most cells of multicellular organisms

What is an Okazaki Fragment, how is it generated, and how does it relate to the lagging strand?

A short segment of DNA synthesized on the lagging strand during DNA replication.

It is generated because DNA polymerase can only synthesize DNA in the 5′ → 3′ direction. Since the lagging-strand template runs in the opposite orientation relative to fork movement, the strand must be copied discontinuously in short pieces, each starting from a new RNA primer.

What are the steps to fix mis-incorporated bases?

1. A wrong nucleotide is inserted.

2. The mismatch disrupts base pairing, so polymerase stalls.

3. The 3′ end of the new strand shifts from the polymerase active site to the 3′→5′ exonuclease site.

4. The incorrect nucleotide is excised.

5. The corrected 3′ end moves back to the polymerase site.

6. The correct nucleotide is inserted.

7. DNA synthesis resumes.

What are the steps for telomere lengthening?

1. Telomerase binds to the 3′ overhang at the end of the chromosome.

2. The telomerase RNA base-pairs with the telomere DNA and serves as a template.

3. Telomerase extends the 3′ end by adding telomere repeat sequences.

4. Telomerase translocates and repeats the extension process to add more repeats.

5. Primase lays down an RNA primer on the complementary strand.

6. DNA polymerase fills in the complementary DNA strand.

7. The RNA primer is removed and replaced with DNA as much as possible.

8. Ligase seals the remaining nick, leaving a short 3′ overhang that protects chromosome ends.

Name and define the parts of the ribonucleo protein telomerase.

TERT (telomerase reverse transcriptase)

The protein catalytic subunit

Functions as a reverse transcriptase that adds DNA telomere repeats to the 3′ end of chromosomes

TERC / TR (telomerase RNA component)

The RNA subunit

Contains the template sequence that telomerase uses to synthesize telomeric DNA repeats

What are the five DNA Polymerases of E. coli and their function?

Pol I = Okazaki fragment processing and DNA repair
Pol II = Translesion synthesis

Pol III = Chromosome replication

Pol IV = Translesion synthesis

Pol V = Translesion synthesis

Explain the steps for: given a DNA sequence and the telomerase, be able to add nucleotide repeats to the telomere on paper

1.) Find the 3′ end of the DNA strand being extended.

Telomerase adds repeats to the 3′ end of the telomere.

2.) Look at the telomerase RNA template.

This RNA is complementary to the DNA repeat that will be added.

3.) Base-pair the telomerase RNA with the existing telomere end.

Match the RNA template to the last few DNA bases at the 3′ overhang.

4.) Add DNA nucleotides complementary to the RNA template.

Use base-pairing rules:

5.) Extend the DNA in the 5′ → 3′ direction.

Add the new bases onto the 3′ end of the DNA strand.

6.) Translocate telomerase and repeat.

After one repeat is added, shift telomerase so its RNA template pairs again with the new end, then add another repeat.

What is the fundamental idea of Transcription?

Transcription of DNA into RNA. The DNA duplex opens to allow a complementary RNA copy to be made from one DNA strand (the template). Synthesis proceeds in the 5′→3′ direction in the growing RNA strand.

What is the chemical mechanism of RNA synthesis?

The addition of an rNTP to a growing transcript is a Mg2+ -dependent reaction that produces a 5′→3′ phosphodiester linkage.

The step-wise process of Transcription had three distinct phases for generating mRNA:

1. Initiation– regulated by protein complexes forming at the promoter

2. Elongation- efficient phase of RNA polymerase extension through

   the gene coding sequence

3. Termination- a signal in the DNA halts the RNA polymerase movement down the template, so enzyme is released

Outline the steps of transcription including initiation, elongation and termination. Use figure 15.7 to describe the steps.

1. Binding of RNA polymerase core to the DNA promoter

2. Formation of transcription bubble

3. Initiation

4. Elongation (promoter clearance)

5. Termination and Recycling

What are the basic outline steps of Transcription Initiation?

1. Formation of transcription preinitiation complex (PIC)

2. Melting of DNA strands

3. Synthesis of RNA oligomers

4. Phosphorylation RNA polymerase II CTD (C-terminal domain) and conformation change

5. Release from “general transcription factors”= promoter clearance.

What is the role of Transcription PreInitiation Complex (PIC) in transcription initiation?

The Transcription Pre-Initiation Complex (PIC) assembles at the promoter and positions RNA polymerase II correctly at the transcription start site so transcription can begin.

Its role is to:

recognize the promoter

recruit and position RNA polymerase II

help unwind the DNA

prepare the polymerase to start RNA synthesis

What is the Transcription PreInitiation Complex (PIC) made up of?

RNAp (RNA polymerase)+ TFII’s (Transcription Factor of Polymerase II) = PIC

What do Basal Transcription factors do?

bind the promoter and help the RNA polymerase (RNAp) find the transcription start site (TSS).

What is TBP and is its deal?

TATA-box binding protein

TBP is found in the TFIID

TBP binds a TATA box cis element
It is a monomer with a saddle-shaped structure; the two halves show an overall dyad symmetry but are not identical (somewhat reflective but not a mirror image).

Interacts with multiple transcription factors (TFs, TFIIB and TFIIA).

Binds in the minor groove and significantly bends DNA.

TBP binds the promoter and helps assemble the transcription machinery at the correct start site.

What is the TATA box and its role in Transcription?

The TATA box is a promoter DNA sequence that helps mark where transcription should begin. Its role is to provide a binding site for TBP (part of TFIID), which helps recruit and position the pre-initiation complex and RNA polymerase II at the promoter.

What are TFIID, TFIIB and TFIIA?

Transcription Factors (TFs)

What are the steps in the formation of the PIC and subsequent events?

1)TFIID is the first to interact (contains TBP)

2)TFIIA joins =stabilize TBP w/ DNA

3)Followed by TFIIB=bridges TFs with DNA

4)TFIIF joins with RNA pol II

5)Other TFII proteins join:

a)TFIIE= modulates F

b)TFIIH=helicase=unwinds the DNA (helicase) and phosphorylates CTD (kinase)

6)Phosphorylation of CTD→ PoL II Conformational Change (Allosteric Regulation)

7)Release of the PoL II from the Promoter

Explain this statement, "Large Multi-Protein Complex Regulate Transcription Initiation"

It means that transcription initiation is not controlled by just one protein, but by many proteins working together as a large complex at a gene’s promoter and regulatory DNA.

What do activators do?

These proteins bind to genes at sites known as enhancers and speed the rate of transcription.

What do repressors do?

These proteins bind to selected sets of genes at sites known as silencers and thus slow transcription

What do coactivators do?

These “adapter” molecules integrate signals from activators and perhaps repressors

What do Enhancers do?

Enhancers are regulatory DNA sequences that increase transcription of a gene. They work by binding activator proteins, which help recruit or stabilize the transcription machinery at the promoter, often through DNA looping.

What do silencers do?

Silencers are regulatory DNA sequences that decrease or repress transcription. They work by binding repressor proteins, which interfere with the transcription machinery or recruit factors that make the DNA less transcriptionally active.

What do basal transcription factors do?

In response to injuctions from activators, these factors position RNA polymerase at the start of transcription and initiate the transcription process

What do cell Specific Transcription Factors do?

Determines Gene / Cell Program and the specified transcription

Describe the concept of the transcriptome and how it plays a role in defining a cell/tissue/etc type.

The transcriptome is the set of RNAs expressed in a cell, and it defines cell/tissue identity by reflecting which genes are active and therefore what functions that cell can perform.

What happens during Transcription Elongation

Phosphorylation of the CTD and RNA polymerase conformation change →allows it to move away from the promoter: Promoter Clearance.

Explain the roles of the CTD and how it functions to carry out said functions.

The CTD (C-terminal domain) is the tail of RNA polymerase II that acts as a platform for coordinating transcription and RNA processing.

Main roles of the CTD

Helps regulate the transition from initiation to elongation

Recruits RNA-processing factors

Coordinates capping, splicing, and polyadenylation

Links transcription with RNA maturation

How it functions

The CTD works through reversible phosphorylation of its repeated amino-acid sequence.

Different phosphorylation patterns at different stages of transcription

These patterns allow different proteins to bind the CTD at the right time

As RNA polymerase moves along the gene, the CTD helps bring in:

5′ capping enzymes early

splicing factors during elongation

3′ end cleavage and polyadenylation factors near termination

Define promoter clearance/escape in the eukaryotic system

Promoter clearance (promoter escape) in eukaryotes is the step where RNA polymerase II successfully breaks away from the promoter and the pre-initiation complex after making the first short RNA nucleotides and transitions into productive elongation.

Explain what the closed and open complex of Transcription

Closed complex: the stage of transcription initiation where RNA polymerase and transcription factors are bound to the promoter, but the DNA is still double-stranded and not yet unwound.

Open complex: the stage where the DNA at the promoter has been locally unwound, exposing the template strand so RNA synthesis can begin.

Explain the closed and open complex of RNA polymerase

Closed complex: RNA polymerase is bound to the promoter, but the DNA is still double-stranded and has not been unwound yet.

Open complex: RNA polymerase has locally unwound the DNA at the promoter, exposing the template strand so transcription can begin.

Explain the conversion of the closed complex to the open complex by sigma factors.

(a) σ70 induces strand opening and formation of the open complex, without the need for ATP or other factors. All other sigma factors except σ54 function in the same way. (b) σ54 requires an AAA+ protein to form the open complex at a promoter. The AAA+ protein is a hexamer that binds an upstream activator sequence (UAS), then also binds σ54 in the closed complex, creating a DNA loop. Such DNA looping is often facilitated by auxiliary DNA-binding and bending proteins such as integration host factor (IHF). AAA+ hydrolyzes a bound ATP, using the favorable energetics of this reaction to drive opening of the complex.

What generally happens during Transcription Termination?

Eukaryote elongation and termination are coupled to mRNA processing

Eukaryotic mRNA is modified at the 5 ́ and 3 ́ ends and internally (cap, splicing and polyadenylation)

Factors (proteins) bind to these features to carry out Termination

What happens during Eukaryotic Primary Transcription Termination and the steps?

Consensus nucleotide sequences signal for mRNA cleavage and polyadenylation

1. Hexamer AAUAAA is bound by Cleavage and Polyadenylation Specificity Factor (CPSF)

2. Cleavage stimulating factor (CstF) binds G-U rich element beyond cleavage site

Describe the steps and molecules involved in mRNA cleavage and subsequent polyadenylation.

1.) RNA polymerase II transcribes the polyadenylation region of the pre-mRNA.

2.) The AAUAAA polyadenylation signal is recognized by CPSF.

3.) A GU/U-rich downstream sequence element is recognized by CstF.

4.) CFIm and CFIIm help assemble the 3′-end processing complex.

5.) The pre-mRNA is cleaved at the cleavage site, usually near a CA sequence, by CPSF73.

6.) Poly(A) polymerase (PAP) adds a string of adenines to the new 3′ end without using a template.

7.) PABPN1 binds the growing poly(A) tail, stimulates further tail addition, and helps control tail length.

Key molecules

cis elements: AAUAAA, cleavage site, GU/U-rich downstream element

proteins: CPSF, CstF, CFIm, CFIIm, CPSF73, PAP, PABPN1

What is the first half of steps for generating the 3 ́-end of the mRNA?

1. CPSF and CstF travel with the RNA polymerase II tail

2. Transferred to 3’-end of transcript (processing consensus sequence) as it emerges from polymerase

3. Additional proteins assemble and RNA is cleaved

4. Cleaving stimulates Polyadenylation

What is the second half of steps for generating the 3’-end of the mRNA (Polyadenylation)?

Poly-A polymerase (PAP) adds approximately 200 A nucleotides to the 3’ end

CPSF (Cleaving AND PolyA factor) disassociates.

PAP also disassociates.

Poly-A-binding proteins (PABPs) bind to growing poly-A tail. As soon as A’s are added and continue to associate.

Poly(A)-binding protein promotes export from the nucleus and translation, and inhibits degradation.

Fill in the blank
Polyadenylation is ____

template-independent

Explain the process and components (proteins and sequences) involved in transcription termination

Transcription termination is the process that stops RNA synthesis and causes RNA polymerase to disengage from the DNA and release the RNA transcript.

In eukaryotes (RNA polymerase II)

1. RNA polymerase transcribes the polyadenylation signal in the RNA, usually AAUAAA.

2. Proteins that recognize this sequence bind, including CPSF and CstF, along with other cleavage factors.

3. The RNA is cut downstream of the polyadenylation signal.

4. Poly(A) polymerase adds the poly-A tail to the released RNA.

5. The leftover RNA still attached to polymerase is degraded by a 5′→3′ exonuclease such as Xrn2, which helps knock polymerase off the DNA and terminate transcription.

Explain the process of Polyadenylation include the proteins, cis elements and steps.

Polyadenylation is the 3′-end processing step where a pre-mRNA is cleaved and then a poly(A) tail is added to its 3′ end. This helps make the mRNA more stable and ready for export and translation.

Key cis elements

• AAUAAA polyadenylation signal, usually 10–30 nt upstream of the cleavage site

• often a CA cleavage site

• a GU/U-rich downstream sequence element (DSE)

• often an upstream UGUA element that helps factor binding

Key proteins

• CPSF binds the polyadenylation signal; CPSF73 is the endonuclease

• CstF binds the downstream GU/U-rich element

• CFIm and CFIIm help assemble/activate processing

• PAP = poly(A) polymerase, which adds the A tail

• PABPN1 binds the growing tail and helps control tail length

Concise steps

1. The AAUAAA signal and nearby 3′-end elements are transcribed.

2. CPSF, CstF, CFIm, and CFIIm assemble on these cis elements.

3. The pre-mRNA is cleaved at the cleavage site by CPSF73.

4. PAP adds adenines to the new 3′ end without a template.

5. PABPN1 binds the tail, stimulates extension, and helps set tail length.

Provide a formula and tips for interpreting EMSA data

Ask these in order:

Is there a shifted band?

→ If yes, protein binds probe.

Does specific competitor remove the shift?

→ If yes, binding is sequence-specific.

Does mutant/nonspecific competitor fail to remove the shift?

→ If yes, this strengthens specificity.

Does the correct antibody cause a supershift or disrupt the shift?

→ If yes, this supports the identity of the protein.

One-line meanings of common EMSA lanes
Probe only → free probe position

Probe + protein → tells you whether binding occurs

+ specific competitor → tests specificity

+ mutant competitor → tests whether exact sequence matters

+ nonspecific competitor → tests whether binding is just random

+ antibody → helps identify the binding protein

Mutant probe → tests whether the binding site itself is required

What is the role of the antibody in the EMSA method?

The antibody is used to help identify the specific protein bound to the DNA probe.
It does this by:

causing a supershift if it binds the protein-DNA complex, making the complex move even more slowly on the gel

or sometimes blocking/disrupting binding, which makes the shifted band weaken or disappear

So, the antibody’s role is to confirm the identity of the DNA-binding protein in the shifted complex.

What is the role of the probe in the EMSA method?

the probe is the labeled DNA fragment that contains the suspected protein-binding site.

Its role is to:

provide the DNA sequence the protein may bind to

allow you to detect binding on the gel

show a shifted band if a protein binds it

What is the role of the “shift” in the EMSA method?

The shift is the slower-moving band that appears when a protein binds the DNA probe.

Its role is to show that:

a DNA–protein complex formed

the protein-bound probe moves more slowly than the free probe because it is larger

So, the shift is the evidence of binding between the protein and the DNA sequence.

What is the role of the super shift in the EMSA method?

the supershift is an even slower-moving band caused when an antibody binds to the protein-DNA complex.

Its role is to:

help identify the specific DNA-binding protein

provide stronger evidence that the shifted complex contains the suspected protein

So, the supershift is used to confirm protein identity in the DNA-protein complex.

What is the role of the specific competitor in the EMSA method?

The specific competitor is an unlabeled DNA fragment with the same sequence as the labeled probe.

Its role is to:

compete with the labeled probe for protein binding

test whether the binding is sequence-specific

If adding the specific competitor reduces or eliminates the shifted band, that shows the protein is specifically binding that DNA sequence.

What is the role of the mutant competitor in the EMSA method?

The mutant competitor is an unlabeled DNA fragment with a mutated binding site.

Its role is to test whether the protein requires the correct specific DNA sequence for binding. Because the binding site is altered, the mutant competitor usually does not compete well for the protein.

So:

If the specific competitor removes the shift but the mutant competitor does not, that shows the binding is sequence-specific

If the mutant competitor also removes the shift, the binding may be less specific than expected

What are the controls in the EMSA method and what do they control for?

Probe-only control

Shows the position of the free probe and controls for probe migration without protein.

Probe + protein/extract lane

Shows whether a shift occurs, meaning binding happened.

Specific competitor control

Uses unlabeled DNA with the same sequence as the probe. Controls for sequence-specific binding. If the shift decreases, binding is specific.

Mutant competitor control

Uses unlabeled DNA with a mutated binding site. Controls for whether the exact binding sequence is required.

Nonspecific competitor control

Uses unrelated DNA. Controls for nonspecific DNA binding.

Antibody / supershift control

Controls for the identity of the DNA-binding protein. A supershift or disrupted shift supports that the suspected protein is in the complex.

Irrelevant antibody control

Controls for nonspecific effects of adding antibody.

How does one transcribe an mRNA provided the DNA sequence?

1.) Identify the template strand

RNA polymerase reads the template DNA strand in the 3′ → 5′ direction.

2.) Build the RNA strand antiparallel to it

The mRNA is made in the 5′ → 3′ direction.

3.) Use base-pairing rules

DNA A → RNA U

DNA T → RNA A

DNA C → RNA G

DNA G → RNA C

4.) If you are given the coding strand instead

The mRNA sequence is the same as the coding strand, except you replace T with U.

Quick shortcut

Template strand → make the complement

Coding strand → copy it and change T to U

Identify and define the template and non-template strand

Template strand: the DNA strand that RNA polymerase reads during transcription. It is read 3′ → 5′ so the RNA can be made 5′ → 3′. The mRNA is complementary to this strand.

Non-template strand (coding strand): the DNA strand that is not read by RNA polymerase. It is also called the coding strand because its sequence matches the mRNA except that DNA has T and RNA has U.

Concisely define and identify polymerases in terms of their dependence, template, product.

DNA polymerase = DNA-dependent DNA polymerase

uses DNA as the template

makes DNA

RNA polymerase = DNA-dependent RNA polymerase

uses DNA as the template

makes RNA

Reverse transcriptase = RNA-dependent DNA polymerase

uses RNA as the template

makes DNA

RNA-dependent RNA polymerase = RNA-dependent RNA polymerase

uses RNA as the template

makes RNA

Outline the chemical process that occurs in the process of transcribing a nucleotide

During transcription, an incoming ribonucleoside triphosphate (rNTP) base-pairs with the complementary DNA template base. RNA polymerase then catalyzes a reaction in which the 3′ OH of the growing RNA strand attacks the α-phosphate of the incoming rNTP. This forms a phosphodiester bond and releases pyrophosphate (PPi). The RNA strand is therefore extended by one nucleotide in the 5′ → 3′ direction.