Chapter 13 - Transcription

Question 1

What is the structure of RNA?

Answer 1

Single strand of nucleotides, sugars, and phosphates

2’ carbon has hydroxy instead of oxygen, so it is less stable than DNA

Can form secondary structures like loops and hairpins because it’s single stranded - plays a role in gene expression

Joined by phosphodiester bonds

Question 2

What are the 3 components of transcription?

Answer 2

Template - all RNA synthesized from DNA

The substrate (rNTPs - ribonucleic triphosphates)

The transcription apparatus

Question 3

What are the key points of the template for transcription?

Answer 3

Transcription only takes place on one strand of DNA (for a single gene) - this is the template strand
- Other strand is the non-template strand

RNA molecule synthesized is both complementary and antiparallel to the template strand of DNA
- Has the same polarity and bases (other than U/T) as the non-template strand

While a single gene is transcribed from a single DNA strand, different genes can be transcribed from different strands (either strand can act as template, depending on the gene)

Core sequences necessary for transcription - known as transcription unit

Question 4

What are the 3 sequences in the transcription unit?

Answer 4

Promoter
- DNA sequence that transcription apparatus recognizes and binds to
- Indicates which of the two strands is going to be template strand
- Also determiens direction of transcription and where transcription start site it (where first nucleotide is laid down)
- Almost always just upstream of transcription start site

RNA-coding region
- Sequence of DNA nucleotides that is copied into the RNA molecule

Terminator
- Sequence of nucleotides that signals where transcription is going to end
- Transcription only ends when terminator site is transcribed into RNA molecule

Question 5

What are the key points for the substrate for transcription?

Answer 5

Nucleotides are added at 3’ OH when two phosphates are cleaved and a phosphodiester bond is formed between the cleavage of the phosphate and 3’ OH of next sugar

During RNA synthesis, nucleotides are always added to the 3’ end of the synthesis strand

RNA synthesis does not require primers for nucleotides because it already has OH

Question 6

What are the key points of the transcription apparatus for transcription?

Answer 6

In prokaryotes:
- 2 alpha subunits, 1 beta, 1 beta’, and optional omega (omega helps stabilize RNA polymerase)
- One enzyme, sigma factor, controls binding of polymerase to promoter
- After binding, sigma factor is removed from polymerase

In eukaryotes:
- Multiple RNA polymerases (I-V) that transcribe different types of RNA
- RNA polymerase II transcribes pre-mRNA

Question 7

What is a holoenzyme?

Answer 7

RNA polymerase subunits + sigma factor (in prokaryotes)

Question 8

What is the upstream region?

Answer 8

Direction opposite of transcription (left of transcription start site)

First nucleotide is -1

Question 9

What is the downstream region?

Answer 9

With direction of transcription (right of transcription start site)

First nucleotide is +1

Question 10

What are the 3 steps of transcription in bacteria?

Answer 10

Initiation, elongation, termination

Question 11

What are the 4 steps of initiation in bacterial transcription?

Answer 11

1. Requires transcription apparatus to recognize and bind to the promoter
2. Form transcription bubble
3. Form first bonds between template and rNTPs
4. Escape of the transcription apparatus from the promoter

Question 12

What happens in step 1 of initiation of transcription in bacteria?

Requires transcription apparatus to recognize and bind to promoter

Answer 12

Binding is where the selectivity of transcription is highly enforced - where it binds determines what parts are transcribed

Sigma factor and RNA polymerase will form holoenzyme (transcription apparatus) and bind to consensus sequence

Question 13

What are consensus sequences in prokaryotes?

Answer 13

Short stretches of nucleotides that are common to prokaryotic promoters

Location within promoter is similar

Orientation and location of consensus sequence determines which strand is template strand

Most common is -10 sequence - called Pribnow box (TATAAT)
- Won’t all be the exact sequence, but they most commonly are

Other common is -35 consensus sequence - TTGACA

Mutations at consensus sequences can change the rate of transcription by changing the affinity for RNA polymerase and promoter sequence
- Up mutations = speed up
- Down mutations = slow down

Question 14

What happens in step 2 of initiation of transcription in bacteria?

Form transcription bubble

Answer 14

Transcription apparatus binds weakly to promoter region

Goes through conformation change and binds more tightly to region, unwinding part of the DNA

Transcription bubble begins at -10 consensus sequence and extends for about 14 nucleotides
- Includes -10 sequence and transcription start site in bubble

Question 15

What occurs in step 3 of transcription in bacteria?

Form first bonds between template and rNTPs

Answer 15

RNA polymerase starts binding complementary rNTPs to template strand with no primer required

Binds 2 of 3 phosphate group on next rNTP to be cleaved as it’s added to 3’ end
- Initial rNTP does not lose its triphosphate on 5’ end

Question 16

What happens during step 4 of initiation of transcription in bacteria?

Escape of transcription apparatus from the promoter

Answer 16

Often during initiation there is formation and release of short transcription sequences while RNA polymerase is still on promoter region - called abortive initiation
- Conformational change in promoter occurs that allows abortive initiation to occur

Then, RNA polymerase generates a long enough strand that it takes off down DNA template and transcribed entire strand

Occurs while holoenzyme is still bound to promoter

Signals start of elongation

Question 17

What occurs during elongation for transcription in bacteria?

Answer 17

Abortive initiation (last step in initiation) signals start of elongation

RNA polymerase continues unwinding DNA at downstream end, adding nucleotides to 3’ end, and rewinding DNA at upstream end

During elongation, transcription can pause - tends to be a slower process than DNA replication
- Pauses help correlate transcription and translation
- Also helps with RNA processing in eukaryotes

RNA polymerase is highly accurate because it has proofreading capabilities while it synthesizes RNA

Question 18

What occurs during termination for transcription in bacteria?

Answer 18

RNA polymerase elongates new RNA molecule until it transcribes terminator sequence - doesn’t stop at termination site, only after it transcribes it

RNA polymerase stops synthesis

RNA molecule releases from polymerase

RNA molecule dissociates from template

RNA polymerase detaches from template

Question 19

What are the two types of terminations in transcription in bacteria?

Answer 19

Rho-dependent termination and rho-independent termination

Question 20

What occurs in rho-dependent termination?

Answer 20

Rho- dependent terminator necessitates the presence of a protein called the rho factor to terminate transcription

Terminator sequence on DNA template causes RNA polymerase to pause when it hits it

Upstream of terminator, DNA sequence encodes for rho utilization site (rut site) in the RNA
- Rut site serves as binding site for rho factor

Rho factor binds in, follows RNA polymerase as it is synthesizing RNA in 3’ direction until RNA polymerase pauses at termination site
- At this point, rho factor catches up to polymerase
- Rho factor has helicase activity in it, so it aids in unwinding RNA strand from DNA strand
- At this time, transcription ends

Question 21

What occurs in rho-independent termination?

Answer 21

No rho factor is needed

Sequence contains inverted repeats, which causes hairpin to form on synthesizing RNA molecule

String of adenines transcribes to string of uracils, which causes RNA polymerase to pause there

Hairpin causes RNA instability and allows it to separate from DNA template, which ends transcription

Question 22

What occurs before transcription in eukaryotes begins?

Answer 22

Modification of chromatin structure of DNA so proteins necessary to trasncription can access the DNA

Occurs in form of chromatin remodeling, histone changes, etc.

Question 23

What are transcription factors (TFs)?

Answer 23

Accessory proteins that recruit polymerases to promoter regions

Affect level at which transcription can occur through its recruitment of polymerases

Question 24

What is the basal transcription apparatus?

Answer 24

General transcription factors and RNA polymerase combining together

Binds into promoter region near start site, which is sometimes enough to initiate transcription

Question 25

What are the two parts of promoters for RNA polymerase II in eukaryotes?

Answer 25

Core promoter

Regulatory promoter

Question 26

What is the core promoter?

Answer 26

Located immediately upstream of gene

Where basal transcription apparatus binds to

Typically includes one or a few consensus sequences
- Most common is TATA box (string of TATAs) at -25

Question 27

What are regulatory promoters?

Answer 27

Located immediately upstream from core promoter

Transcription factors bind to transcription sequences on promoter to directly or indirectly make contact with basal transcription apparatus to affect the rate at which transcription occurs

Transcription factors can also bind to enhancer regions in regulatory promoters

Question 28

What are the three steps of transcription in eukaryotes?

Answer 28

Initiation, elongation, termination

Question 29

What occurs during initiation of transcription in eukaryotes?

Answer 29

Assembly of transcription machinery on core promoter region
- Includes RNA polymerase II and several transcription factors that form giant complex of polypeptides (50+ proteins)

Regulatory proteins that bind to promoter that alter chromatin structure - opens DNA for transcription to occur
- Also recruit basal transcription apparatus to the promoter region

Basal transcription apparatus includes RNA polymerase II and several transcription factors
- TFIIA, B, D (recognizes and binds to TATA box), E, F, and H - serve same function as sigma factor in prokaryotes
- Recruit more proteins called TATA binding proteins that enter minor groove of DNA to bend and unwind DNA (creates transcription bubble)

More transcription factors will bind and position RNA polymerase over transcription start site
- DNA strand positioned within active site of RNA polymerase - where DNA is opened is the open complex
- Once open complex is formed, abortive initiation occurs until elongation starts

Question 30

What occurs during elongation in transcription in eukaryotes?

Answer 30

After about 30 nucleotides are added, RNA polymerase begins to move downstream of start site, and elongation starts

Transcription factors are left behind, which allows them to reinitiate transcription if they need to as they can recruit another polymerase

DNA is unwound in transcription bubble

RNA and DNA hybrid is formed at active site - where RNA polymerase is adding RNA nucleotides to strand

Newly synthesized RNA and DNA exists and is rewound in the process

Question 31

What occurs during termination in transcription in bacteria?

Answer 31

RNA polymerase I - uses something similar to rho-dependent terminator

RNA polymerase III - uses something similar to rho-independent terminator

RNA polymerase II - doesn’t have specific sequence at which termination occurs
- Continues to synthesize RNA for hundred of thousands of bases past the coding region
- Pre-mRNA is cleaved at a specific sequence of RNA molecule as transcription is still taking place
- Cleavage cuts pre-mRNA into two pieces - mRNA strand and RNA tail that sticks out of polymerase
- Protein with exonuclease activity attaches the 5’ end of the tail coming out of the polymerase and moves toward 3’ end, chewing up RNA that is left
- When it reaches transcription apparatus, it stops transcription