HRR: transcription I

Question 1

___ polymerases have a proofreading function, ___ do not

Answer 1

DNA; RNA

Question 2

What direction in RNA polymerase reading DNA? What direction does it synthesize?

Answer 2

Reads 3’ to 5’, synthesizes 5’ to 3’….always!

Question 3

What is denovo synthesis?

Answer 3

RNA Polymerase initiates RNA synthesis de novo by joining 2 ribonucleotides together to form the first 3’5’ phosphodiester bond. Unlike DNA synthesis, a primer is not required

Question 4

RNA is synthesized _’ to _’, making bonds _’ to _’

Answer 4

5 to 3; 3 to 5

Question 5

Specify the protein subunits of prokaryotic RNA Polymerases. Explain how the
sigma (s) subunit is directed to specific promoters to initiate transcription.

Answer 5

Prokaryotes have one type of RNA polymerases, with various subunits. There is an alpha, beta, beta prime, and omega. The beta subunits bind to the DNA and have a helicase to unwind the DNA and the catalytic site to catalyze bond formation.

The sigma subunit is needed to recognize where the promoter is; it looks for the specific sequence and recognizes it, allowing the polymerase to bind in order to begin transcription. There tends to be two consensus sequences

Question 6

in RNA polymerase in prokaryotes, which subunits make the core enzyme? what makes it a holoenzyme?

Answer 6

a, b, b’; sigma

Question 7

Define promoter specificity in transcription of prokaryotes

Answer 7

Only one RNA Polymerase exists in prokaryotes. However, the RNA Polymerase holoenzyme can initiate transcription of all specific genes via the sigma subunit

Question 8

What is the main enzyme used in transcription of DNA?

Answer 8

RNA polymerase; it is a processive enzyme, meaning it stays on the strand instead of dissociating

Question 9

Differentiate between a closed and open promoter complex during transcription in prokaryotes

Answer 9

Closed: the polymerase has bound to the DNA, but has not unwound the strands yet.

Open: the polymerase has begun to unwind the DNA, forming the open promoter complex.

Question 10

describe initiation of transcription in prokaryotes

Answer 10

RNA polymerase looks for the promoter site by sliding along the DNA (it has low affinity for other sequences besides the promoters, giving it the ability to do this). The sigma subunit binds to the promoter sequences. The polymerase then begins to unwind the DNA to form an open promoter complex. It can then form the first phosphodiester bond. The sigma subunit then releases from the polymerase.

Question 11

describe elongation during transcription in prokaryotes

Answer 11

the core RNA moves the transcription bubble along the chromosome. A type II topoisomerase called gyrase is used to create negative supercoils ahead of the bubble to prevent positive supercoiling. A type I topoisomerase is used to relax negative supercoils behind the bubble.

Question 12

describe termination in transcription of prokaryotes

Answer 12

-Template directed: a GC rich repeat forms a hairpin, halting the transcription reaction. Following the hairpin is a series of U bases, making for weak binding between the strand and they polymerase. These two factors cause the RNA polymerase to dissociate from the template.

-Rho-directed: rho protein Rho protein binds to specific sequences in newly synthesized RNA. Rho protein has ATP-dependent helicase activity that
dissociates RNA from template strand

Question 13

what does type I RNA polymerase make

Answer 13

produces 45s pre-ribosomal RNA; found in nucleolus specifically

Question 14

what does type II RNA polymerase make

Answer 14

Type II: produces pre mRNA, primary miRNA, snRNA, lncRNA

Question 15

what does type III RNA polymerase make

Answer 15

Type III: produces tRNA, 5s rRNA, some snRNA
mitochondrial: all mitochondrial RNAs

Question 16

Describe the basic components required for eukaryotic RNA Polymerases to initiate
transcription of a gene.

Answer 16

Core RNA polymerase: mundane work; comprised of multiple protein subunits
General transcription factors: required for basal transcription of all genes
Specific transcription factors:
-Activators/repressors: they activate/repress transcription of a specific gene. They bind directly to cis-acting DNA sequences such as enhancers or silencers. They recruit co-regulators.
-Co-regulators: they don’t bind directly to DNA, they instead bind to activators or repressors.

Question 17

Differentiate between cis-acting elements and trans-acting factors that regulate transcription of individual genes

Answer 17

Cis acting: DNA sequences in the gene itself that regulate it; they are part of the gene. Examples include the core promoter, enhancers, silencers
Trans acting: they come from genes other than the target gene, but they regulate the target. Examples include specific transcription factors.

Question 18

Describe the basic components required to form a transcriptionally-active RNA Polymerase I complex and how it functions in the production of ribosomes.

Answer 18

Core RNA: 10-12 subunits; binds to the promoter region

Selectivity factor 1: trans-acting element; 4 general transcription factors that binds to DNA sequence in the core promoter of rRNA gene

Upstream binding factor: a specific transcription factor for RNA polymerase I. it’ll bring in other co-regulators and interact with SL1 to promote assembly of an active polymerase

Question 19

describe rRNA genes

Answer 19

A human diploid cell contains approximately 400 rRNA genes arranged in tandem repeats along the 5 acrocentric chromosomes [13, 14,15, 21, 22] in the nucleolus. Each gene for rRNA is separated by spacer (non transcribed) DNA

Question 20

Describe the main types of sequence elements in the core promoter of a gene and their
corresponding functions in initiation of transcription by RNA Polymerase II.

Answer 20

Core promoter: minimal stretch of DNA that is sufficient to direct initiation of transcription. They have several consensus sequence elements.
-Initiator, tata box, motif 10 elements, downstream promoter element, BRE

Question 21

Describe the main components of a pre-initiation complex (PIC) and how they are assembled on the core promoter of all eukaryotic genes.

Answer 21

Core promoter: multimeric enzyme with a c-terminal tail domain containing serine residues that can be phosphorylated.

General transcription factors: TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs).

Mediator complex: It functions in transducing signals from specific transcription factors bound to cis-acting elements to components of the PIC that assemble on the core promoter

Question 22

what is the primary method of regulating transcription for mRNA using RNA polymerase II?

Answer 22

regulating the assembly of the primary initiation complex

Question 23

Describe initiation with RNA polymerase II in eukaryotes

Answer 23

TFIID (basal transcription factor) directs assembly and helps identify the transcription start site

TFIIH: has multiple subunits and 2 main functions:
-Helicase activity: unwinding DNA strands in the core promoter
-Protein kinase activity: phosphorylating the CTD in RNA polymerase II, facilitating promoter release for elongation to occur

TAFs act like a ruler and positions tata box binding proteins (TBP) properly

Question 24

Describe the role of TFIID

Answer 24

TFIID directs assembly and helps identify the transcription start site

Question 25

Describe the role of TFIIH

Answer 25

TFIIH: has multiple subunits and 2 main functions:
- Helicase activity: unwinding DNA strands in the core promoter
- Protein kinase activity: phosphorylating the C terminal tail domain (CTD) in RNA polymerase II, facilitating promoter release for elongation to occur

Question 26

Describe the role of TAFs

Answer 26

TAFs act like a ruler and positions tata box binding proteins (TBP) properly

Question 27

Describe the role of the C-terminal domain of RNA Polymerase II in the elongation step.

Answer 27

serines on the CTD can be phosphorylated which releases the polymerase core enzyme from the promoter.

Question 28

Explain how the termination step of transcription is linked to polyadenylation of pre-mRNA

Answer 28

once the strand is transcribed, a polyA tail is added to the 3’ end. this protects the pre-mRNA strand.