Midterm 2 Study Material

Question 1

What is different about ribose vs Deoxyribose (be specific).

Answer 1

On the 2’carbon of Deoxyribose, it does NOT have the OH that ribose has, instead it just has a H.

Question 2

(lec. 8)

True or False:
During DNA transcription, the double helix locally DENATURES.

Answer 2

TRUE!

During DNA transcription, the double helix locally Denatures, and then one strand acts as a template strand.

During DNA transcription, the double helix locally denatures, or unwinds, so that the RNA polymerase can access the template strand and synthesize RNA. This separation creates a “transcription bubble” where transcription occurs.

Question 3

(lec. 8)

True or False:
During DNA Transcription, after the alpha helix has been unwound, incoming Deoxynucleotide triphosphates will come in and base-pair with the bases in the DNA template strand.

Answer 3

FALSE.

During DNA Transcription, after the alpha helix has been unwound, incoming ribonucleotide triphosphates will come in and base-pair with the bases in the DNA template strand.

–> During Transcription, this is when RNA is being synthesized using DNA as a template. It’s in DNA replication that we’d have deoxynucleotide triphosphates.

Question 4

(lec. 8)

True or False:

During DNA transcription, RNA polymerase sequentially joins the rNTPs from 3’ to 5’.

Answer 4

FALSE!

During DNA transcription, RNA polymerase sequentially joins the rNTPs from 5’ to 3’.

Extra info:
(RNA polymerase adds nucleotides in the 5’ to 3’ direction because of the way it catalyzes the formation of bonds between nucleotides. Specifically, it connects the 5’ phosphate group of each incoming rNTP to the 3’ hydroxyl (-OH) group of the growing RNA strand. This reaction releases energy, driving the addition of the nucleotide.

The enzyme can’t add nucleotides in the 3’ to 5’ direction because there wouldn’t be a free 3’-OH group for new bond formation, making 5’ to 3’ the only chemically viable direction for RNA synthesis.)

Question 5

(lec. 8)
True or False:

During transcription, polymerization is energetically favoured because the high-energy bond between the alpha and beta phosphates is replaced by a lower energy bond.

Answer 5

TRUE!

~~Extra Info::
[In nucleotide triphosphates (like ATP, GTP, etc.), the alpha (α), beta (β), and gamma (γ) phosphates refer to the three phosphate groups attached to the nucleotide’s ribose or deoxyribose sugar.

The alpha (α) phosphate is the phosphate group directly attached to the sugar.
The beta (β) phosphate is the second phosphate, linked to the alpha phosphate.
The gamma (γ) phosphate is the third phosphate, linked to the beta phosphate.]

Question 6

(lec. 8 related)

Tell me about the building blocks of mRNA before they get added to the strand. What are they?

Answer 6

Before the building block nucleotides for mRNA are added, they are actually NUCLEOTIDE TRI-PHOSPHATES, which is what ATP is.

So, ATP (Adenosine tri-phosphate) is Adenine with 3 phosphates attached to it.
Guanine, Cytosine, Uracil, all come from guanosine triphosphate, cytidine triphosphate.

So, ATP is the energy-carrying form of adenosine, and similarly, GTP, CTP, and UTP are high-energy forms of guanine, cytosine, and uracil nucleotides. When added to RNA, only the alpha phosphate remains attached, while the beta and gamma phosphates are released as pyrophosphate, which drives the reaction forward.

Question 7

(lec 8.)

During transcription, RNA polymerase begins transcription at gene nucleotide designated ____.

Answer 7

RNA polymerase begins transcription at gene nucleotide designated +1.

Question 8

(lec. 8)
True or false:
during transcription, RNA polymerase travels “downstream” toward the 3’ end on the DNA and downstream bases are designated with positive numbers.

Answer 8

FALSE!

during transcription, RNA polymerase travels “downstream” toward the __5’__ end on the DNA and downstream bases are designated with positive numbers

Question 9

(lec. 8)
During transcription, what do we call nucleotides that are upstream of the +1 site?

And what is the +1 site?

Answer 9

During transcription, we refer to nucleotides that are upstream of the +1 site with negative numbers. so, it’s like a number like.

The +1 site is the spot where the RNA polymerase begins transcription.

Question 10

(lec. 8)

During transcription, what kinds of things lie Upstream of the transcription start site?

What is the Transcription start site called?

Answer 10

Things that lie upstream (into the negative nucleotide #’s) include PROMOTER SEQUENCES that RNA polymerase will recognize and bind to before it begins to transcribe at the transcription start site.

The transcription start site is denoted +1 (and all subsequent nucleotides are designated with positive numbers, like a number line).

Question 11

(lec. 8)

During transcription, what do we call the DNA strand that is being transcribed?

And so what do we sometimes call its compliment strand?

Answer 11

The DNA strand that is being transcribed, is the Template Strand.

its compliment is called the non template strand.

Question 12

(lec. 8)

What are the 3 stages of DNA TRANSCRIPTION, with just a brief definition.

Answer 12

1: INITIATION ~ RNA polymerase binds to the DNA where we need to start transcribing, and it denatures the DNA helix around the start site. It CATALYZES the first phosphodiester linkage. (“Denatures” means that it unwinds. Same concept for proteins becoming denatured, they unfold.)

2: Elongation ~ Polymerase moves along the strand as it synthesizes, in the 3’ to 5’ direction along the TEMPLATE strand.
(so, as we know, this means the mRNA is formed in the 5’ to 3’ direction).

3: termination ~ Polymerase recognizes a stop site, releases a completed RNA and dissociates from DNA.

Question 13

(lec. 8)

During DNA transcription, tell me about Initiation.

Answer 13

initiation is the first phase of DNA transcription.
It begins with RNA polymerase binding to the promoter sequence. “Closed complex” (meaning the two strands are still wound).

Polymerase melts the duplex DNA near the transcription start site, which forms the transcription bubble. “Open complex”, meaning the strands are unwound at that spot.

Polymerase catalyzes phosphodiester linkages of the two initial rNTPs.

Question 14

(lec. 8)

During DNA transcription, tell me about Elongation.

Answer 14

Elongation is the second stage of DNA Transcription (after initiation), and it’s where the mRNA strand is getting longer basically.
polymerase advances down the template strand.

The elongation complex is very stable.

speed of elongation is 1000nt/min, so small genes take a few minutes, but big genes can take hours.

Question 15

(lec. 8)

During DNA transcription, tell me about Termination.

Answer 15

Termination is the LAST step of the transcription process, where the RNA polymerase comes to the STOP codon sequence and releases the completed RNA and dissociates from DNA.

Question 16

(lec. 8)

After Termination in DNA transcription, what is the completed RNA molecule called?

Answer 16

The completed RNA molecule is called the Primary transcript.

Question 17

(lec. 8)

which of the following are substrates used by RNA polymerase in the process of transcription?

a) DNA template

b) ribonucleotide triphosphates

c) deoxyribonucleotide triphosphates

d) inorganic phosphate

e) Red Bull TM

Answer 17

b) ribonucleotide triphosphates

Remember: a substrate is a molecule that undergoes a CHANGE in a reaction catalyzed by an enzyme.

Question 18

(lec. 8)

In DNA transcription, what are sigma factors?

Answer 18

In DNA transcription, sigma factors are proteins that help RNA polymerase bind to specific promoter regions on DNA, initiating transcription.

After transcription begins, the sigma factor is released.

The best known one is σ⁷⁰ (sigma-70), which recognizes TTGACA…TATAAT

Note: They are found ONLY in BACTERIA.

Question 19

(lec. 8)
True or false:

In eukaryotes, genes with a common function are often arranged linearly in operons and transcribed together on a single mRNA.

Answer 19

FALSE!

In PROKARYOTES, genes with a common function are often arranged linearly in operons and transcribed together on a single mRNA.

Question 20

(lec. 8)

Explain the way genes that code for a common function are arranged in prokaryotes vs eukaryotes.

Answer 20

In Prokaryotes, genes with a common function are often arranged linearly in operons and transcribed together on a single mRNA.
–> There are very FEW non-coding gaps of DNA in prokaryotic genomes.

In Eukaryotes, genes for a common function are often scattered across multiple chromosomes.
–> So this fact proves to us that co-regulation of genes does not happen as a result of physical linkage or proximity.

Question 21

(lec. 8)

True or False:

In prokaryotes, mRNAs are transcribed directly from DNA.

Answer 21

True!

Question 22

(lec. 8)

If in prokaryotes, mRNAs are transcribed directly from DNA, is there a difference with eukaryotes?

Answer 22

In eukaryotes, the transcripts must go through several processing steps before becoming mRNAs.

Question 23

(lec. 8)

True or false:

In Eukaryotes, elements that regulate transcription can span many kilobases, in either direction from said transcription site.

Answer 23

True!

In eukaryotes, regulatory elements like enhancers, silencers, and other control sequences can be located many kilobases away from the genes they regulate.

Question 23

(lec. 8)

True or false:

The yeast RNA polymerase is much more complex than the bacterial RNA polymerase.

Answer 23

True!

yeast is a eukaryotic cell, and eukaryotes have more complex RNA polymerase.

It contains more subunits and has additional regulatory requirements due to the complex organization of eukaryotic genomes, which involve chromatin structure, multiple transcription factors, and regulatory elements.

Question 24

(lec. 8)

True or false:

Eukaryotic RNA polymerases contain two large subunits and 10-14 smaller subunits, some of which are common between all 3 types of RNA polymerase in eukaryotes, and some of which are specific.

Answer 24

true!

Question 25

(lec. 8)

How many types of RNA polymerase are in eukaryotes?

Which is more complex than a bacteria RNA polymerase?

Answer 25

Eukaryotes have three main RNA polymerases:

RNA Polymerase I – synthesizes rRNA (ribosomal RNA) in the nucleolus.

RNA Polymerase II – synthesizes mRNA (messenger RNA) and snRNA (small nuclear RNA) and is key for transcribing protein-coding genes.

RNA Polymerase III – synthesizes tRNA (transfer RNA) and some small RNAs like 5S rRNA.

ALL OF THEM are more complex than a bacterial RNA polymerase because eukaryotic transcription requires additional regulation and processing, involving more subunits and factors to accommodate chromatin and complex gene regulation.

Question 26

(lec. 8)

Label each statement about Yeast RNA polymerases as True or False:

1) All 3 yeast polymerases have five (5) core subunits homologous to the β, β’, two α, and ω subunits of E. coli RNA polymerase.

2) All 5 polymerases share the same ω-like subunit, and four other common subunits.

3) Each yeast polymerase contains 3-7 unique smaller subunits.

4) RNA polymerase II largest subunit (RPB1) also contains an essential C-terminal domain (CTD).

5) RNA polymerases I and III contain the same two nonidentical α-like subunits, whereas RNA polymerase II contains two other nonidentical α-like subunits.

Answer 26

1) –>TRUE!
All 3 yeast polymerases have five (5) core subunits homologous to the β, β’, two α, and ω subunits of E. coli RNA polymerase.

2) FALSE!
–>There are only 3 different polymerases in eukaryotes, not 5.
Correct: All 3 polymerases share the same ω-like subunit, and four other common subunits.

3) TRUE!
Each yeast polymerase contains 3-7 unique smaller subunits.

4) RNA polymerase II largest subunit (RPB1) also contains an essential C-terminal domain (CTD).

5) RNA polymerases I and III contain the same two nonidentical α-like subunits, whereas RNA polymerase II contains two other nonidentical α-like subunits.

Question 27

(lec. 8)

General transcription factors are required at every RNA Pol II promoter, which are highly conserved in all eukaryotes - elaborate on this ^^.

Answer 27

This just is saying that RNA Polymerase II in eukaryotes will require essentially the same promoter sequence in yeast, all the way to humans, the structure of these promoter sequences is all very similar.

Question 28

(lec. 8)

What is a Carboxy-terminal domain (CTD)?

Answer 28

The Carboxy-terminal domain (CTD) is a region at the end of the largest subunit of RNA Polymerase II.

It is made of protein (amino acids), and in mammals, it is made of about 52 nearly identical repeats of the amino acids Tyr-Ser-Pro-Thre-Ser-Pro-Ser.
(Tyrosine, Serine, Proline, Threonine).

Question 29

(lec. 8)

RNA molecules that initiate transcription have a _______________ CTD

RNA molecules that are actively transcribing have a _______________ CTD.

Answer 29

RNA molecules that initiate transcription have an unphosphorylated CTD.

RNA molecules that are actively transcribing have a Phosyphorylated CTD.

((More info:))
–>RNA molecules that initiate transcription have an unphosphorylated CTD because, at this stage, the CTD is not yet modified, allowing RNA Polymerase II to assemble with general transcription factors at the promoter and start transcription.
–> Once transcription begins, the CTD becomes phosphorylated, which facilitates the recruitment of various processing factors necessary for mRNA processing (like capping, splicing, and polyadenylation). This phosphorylation acts as a switch, signaling that RNA Polymerase II is actively transcribing, enabling it to coordinate the transcription and processing of the growing mRNA transcript.

Question 30

(lec. 8)

What is The Clamp Domain Of RPBI?

Answer 30

The Clamp Domain of RNA Polymerase II (RPB1) is a structural feature that helps the enzyme maintain a stable interaction with the DNA template during transcription. It encircles the DNA, functioning like a clamp to keep the DNA strands together as RNA Polymerase II moves along the template strand. This domain is crucial for ensuring the fidelity and efficiency of transcription, as it helps prevent the dissociation of the enzyme from the DNA and allows the polymerase to transcribe the gene accurately without losing its grip on the DNA.

Question 31

(lec. 8)

During this lecture, why do we keep talking about RNA polymerase II and not I or III?

Answer 31

because RNA polymerase II is the only RNA polymerase that makes mRNA.

–> RNA Pol I does majority rRNA.
–> RNA Pol III does majority tRNA.
Neither does mRNA, only RNA Pol II

Question 32

(lec. 8)

On the Clamp Domain of RPBI:

It is ____ when downstream DNA is inserted into this region of the polymerase.

It swings ___ when the enzyme is in elongation mode, which anchors the polymerase into the downstream double stranded DNA.

Shut position makes enzyme very processive (human Dystrophin DMD gene - 2 million bases transcribed at 1-2kb per minute = 1 day without dissociating.)

Answer 32

It is open when downstream DNA is inserted into this region of the polymerase.
–>{Obviously it needs to be open to let the DNA come in}

It swings Closed when the enzyme is in elongation mode, which anchors the polymerase into the downstream double stranded DNA.
–>{obviously it needs to be closed to clamp on, hence it’s name}

Shut position makes enzyme very processive (human Dystrophin DMD gene - 2 million bases transcribed at 1-2kb per minute = 1 day without dissociating.)

Question 33

(lec. 8)

Tell me about the difference between Transcribing RNA Pol II and free RNA Pol II relating to the clamp domain.

Answer 33

When RNA Polymerase II is actively transcribing, it differs from the inactive form primarily because the clamp domain in the RPB1 subunit moves to cover a gap in the enzyme. This movement traps the template DNA strand and the growing RNA transcript, ensuring they are held securely during transcription.

A magnesium ion (Mg²⁺) is crucial for the reaction that forms the bonds between nucleotides, known as phosphodiester bonds.

Additionally, the RPB2 Wall domain pushes the template DNA to bend before it exits the polymerase. This bending helps maintain the correct positioning of the DNA and RNA, ensuring that transcription occurs efficiently and accurately.

Question 34

(lec. 8)

What Ion is important for the formation of phosphodiester bond formation during transcription? And why?

Answer 34

The important ion for the formation of phosphodiester bonds during transcription is magnesium (Mg²⁺). It plays a critical role because it stabilizes the negative charges on the phosphate groups of the nucleotides, facilitating the reaction that links nucleotides together to form the RNA strand. This stabilization is essential for the catalytic activity of RNA polymerase.

Question 35

(lec. 8)

What is the pre-initiation complex in transcription?

Answer 35

The pre-initiation complex (PIC) in transcription is a group of proteins, including RNA Polymerase II and general transcription factors, that assemble at the promoter region of a gene before transcription begins. This complex is essential for correctly positioning RNA Polymerase II on the DNA and unwinding the DNA strands so that transcription can start. The PIC ensures that the transcription machinery is ready to synthesize RNA accurately.

Question 36

(lec. 8)

How is RNA polymerase able to unwind the DNA strands? Does it have helicase?

Answer 36

RNA polymerase unwinds the DNA strands during transcription without a separate helicase enzyme.

It has built-in helicase-like activity within its structure that allows it to separate the two strands of DNA as it moves along the template strand.

This unwinding creates a transcription bubble, enabling RNA polymerase to access the DNA and synthesize RNA. The energy required for this process comes from the nucleotide triphosphates (NTPs) that are incorporated into the growing RNA strand.

Question 37

(lec. 8)
What does TFIIB recognition mean?

Answer 37

TFIIB recognition refers to the process by which the general transcription factor TFIIB (Transcription Factor IIB) interacts with the promoter region of a gene during the formation of the pre-initiation complex for transcription. After the initial binding of TFIID (Transcription Factor IID), which includes the TATA-binding protein (TBP), to the promoter, TFIIB recognizes specific DNA sequences near the transcription start site.

(more info:)
This recognition is crucial because it helps position RNA Polymerase II correctly at the promoter and is essential for the recruitment of additional transcription factors and RNA Polymerase II itself. TFIIB serves as a bridge between the promoter DNA and the RNA polymerase, ensuring that the transcription machinery is assembled properly for effective gene expression.

Question 38

(lec. 8)

What are the 4 core promoter elements in eukaryotes?

Answer 38

1: BRE ~ (stands for TFIIB Recignition element)
Located just upstream of the TATA box, it is recognized by TFIIB and is important for the recruitment of RNA polymerase II.

2: TATA box ~ A short DNA sequence (about 25-30 base pairs upstream of the transcription start site) that is recognized by the TATA-binding protein (TBP), helping to position RNA polymerase II.

3: Inr ~ (Initiator)
A DNA sequence located at the transcription start site that helps define where transcription begins.

4: DPE ~ (Downstream Promoter Element)
Found downstream of the transcription start site, it plays a role in transcription initiation, especially in TATA-less promoters.

Question 39

(lec. 8 concept)

Define each of the following RNA Pol II promoter sequences:

1) TATA Boxes

2) Initiator Sequences

3) CpG Islands

Answer 39

1) TATA Boxes: These are short, conserved DNA sequences located about 25-30 base pairs upstream of the transcription start site. They play a critical role in positioning RNA polymerase II; however, even a single base change in the TATA box can significantly decrease gene transcription.

2) Initiator Sequences: These sequences are located at the transcription start site and help define where transcription begins. They are described as degenerate sequences, meaning they can vary in their specific nucleotide composition but still fulfill the same functional role in facilitating transcription initiation.

3) CpG Islands: These are regions rich in cytosine and guanine dinucleotides and are often found near promoter regions of genes, playing a role in regulating gene expression.

Question 40

(lec. 8)

Briefly list the 6 steps of eukaryotic Transcription Initiation.

Answer 40

1) TBP (TATA box Binding Protein) binds to the TATA box on DNA.

2) TFIIB (Transcription Factor IIB) binds to both the DNA and the TBP, melting the DNA at the transcription start site, forming the transcription bubble.

3) A pre-formed complex of RNA Pol II & TFIIF binds to the DNA next.
(RNA Pol II does not directly bind to the TATA box.)

4) TFIIE binds next.

5) A complex called TFIIH (with 9 subunits) binds and is a HELICASE which will unwind the DNA.

6) The CTD of the RNA Pol II gets phosphorylated by the kinase subunit of TFIIH.

Question 41

(lec. 8)

(in eukaryotic DNA transcription)
During the step of TBP binding to the TATA box, what specifically happens conformationally with the TBP?

Answer 41

-The conserved C terminal domain of the TBP binds to the MINOR groove of specific DNA sequences rich in A and T, untwisting and sharply bending the double helix.

Extra info:
(Transcription of most eukaryotic genes requires participation of TBP)

Question 42

(lec. 8)
True or False:

RNA Pol I and RNA Pol III have similar transcription initiation processes and require the same general transcription factors.

Answer 42

FALSE!

RNA Pol I and RNA Pol III have similar transcription initiation processes but require different general transcription factors.

Question 43

(lec. 8)

1: Which RNA polymerase transcribes the 45S precursor of 18S, 5.8S, and 28S rRNA from multiple copies of the pre-rRNA gene?

2: Which RNA polymerase transcribes tRNAs, 5s rRNA, U6 splicing snRNA, and additional small stable RNAs of unknown function?

Answer 43

1: RNA Polymerase II.

2: RNA Polymerase III.

Question 44

(lec. 9)

Do rRNAs code for ribosomes, or only act as a component of the ribosome?

Answer 44

rRNAs (ribosomal RNAs) do NOT code for ribosomes; instead, they act as a structural and functional component of the ribosome. rRNAs combine with proteins to form the two subunits of the ribosome, playing a crucial role in protein synthesis by stabilizing the structure and catalyzing the formation of peptide bonds during translation.