Chapter 21 MINE

What is the role of RNAP in E.Coli?

Uses a single type of RNA Polymerase (RNAP), makes RNA in the 5→3 direction, and does not require a primer to begin the process.

How many distinct RNA polymerases are in Eukaryotes?

3

What is the role of RNAP I?

Produces ribosomal RNA (rRNA), making up 80% of the total RNA in a cell.

What is the role of RNAP II?

Makes messenger RNA (mRNA), which serves as template that codes for polypeptides during protein synthesis, accounts for only 5% of cellular RNA

What is the role of RNAP III?

Produces both transfer RNA (tRNA) and certain types of rRNA, make up about 15% of the cell’s RNA

What is tRNA essential for?

Essential for translation because they deliver amino acids to the ribosome.

What does upstream mean?

Refers to DNA sequence located before the transcription start site; bases are assigned negative numbers

What does downstream mean?

Refers to DNA sequences located after the start site, follows the direction of transcription; bases are assigned positive numbers

What does Templae/Non-coding/Antisense/(-)Strand mean?

This is the DNA strand that RNA polymerase physically reads to synthesize a new RNA strand in the 5→3 direction

What is Non-template/Coding/Sense?(+) Strand mean?

This is the DNA strand complementary to the template, its sequence matches the sequence of the RNA transcript, refered as the coding or the sense strand.

What are exons?

These are the expressed regions of the mRNA that code for the final polypeptide sequence

What are introns?

These are the intragenic regions that separate exons, and are removed from the primary transcript and remaining exons are spliced together to form the mature mRNA product.

How is the initiation site numbered?

(+) 1

Is there a base zero?

Nope! Sequence jumps from -1 to +1

How can histones be covalently modified?

They can experience acetylation, methylation, phosphorylation, and/or ubiquitnation.

How does can histones be covalently modified affect gene expression?

Modifications ca either increase or repress transcription depending on the context of the modification. Acetyaltation is associated with activation.

What is BRE^U?

TFIIB recognition element upstream, located at -35

Where is TATA box located?

-30

Where is BRE^J (TFIIB Recognition Element Downstream) located?

-20

Where is Inr (Intiator) located?

+1

Where is MTE (Motif Ten Element) located?

+20

Where is DPE (Downstream Promoter Element) located?

+30

What is the role of TFIIA?

Assists TBP in its bidning and stabilization at the promoter

What is the role of TFIIB?

Factor acts as a bridge, to help recruit the TFIIF-RNAPII complex to the transcription start site

What is TFIID?

This factor is a large comple that incldues the TATA-binding protein ad various associated factors. Role is to recognize and bind specific core promoter elements, specifically help locate the initiation and downstream promotor element while recruiting TFIIB.

What is TBP(TATA-BInding Protein)?

Part of TFIID, it specfically recongnizes and binds to TATA box sequence located upstream at the promoter

What is TFIIE role?

Its priamry role is to recruit TFIIH to the initation complex

What is the role of TFIIF?

It increases RNAPII’s affinitiy for the start site, helps elongate the mRNA product and binds to the non template strand to stabilize the transcription bubble during its formation.

What is the role of TFIIH?

-It acts as a helicase to physically unwind the DNA and open the transcription bubble

-Along with the mediator complex, it helps phosphorylate the C-terminal domain of RNAPII, helps with beginning elongation phase of transcription

What is the role of enhancers?

DNA sequences that serve as binding sites for proteins intended to increase transcriptional activity

What is the role of silencers?

DNA sequences that help regulate transcription by providing binding sites for proteins that decrease activity

What are regulatory proteins?

These proteins bind to DNA sequences that influence RNA polymerase acitivity.

What is the role of activators?

These proteins bind to enhancers and icnrease the ability of RNAP to transcribe a specfic gene.

What is the role of surpressors?

These proteins bind to silencers and decrease the activity of RNAP, effectively turning down or shutting of gene expression.

What is the mediator?

The mediator is a large protein complex that serves as essential link between distal regulatory signals and the core transcriptional machinery

What is the role of the mediator complex?

-It physically connects activator to the transcription complex at the promoter site

-Works along side TFIIH to phosphorylate the terminal domain of RNAPII, allowing for the elongation phase of transcription to begin.

What are the two ways that transcription may be terminated in E.coli?

-Via Intrinsic Termination and Rho-dependent termination

How does intrinsic Temination work?

:RNAP transcribes a region that pulls apart the RNA-DNA

hybrid helix when the RNA base pairs with itself to form a

hairpin structure.

How does Rho-dependent termination work?

Specifically binds a C-rich region on

the transcript and bumps RNAP off the template.

What are operons?

They are functional units of DNA consisting of several genes under the control of a single promoter

How are operons advantageous to prokaryotes?

-Primary advantage is ability to coordinate the expression of related genes:

• Efficient regulation” All necessary proteins are produced simultaneously in response to a single protein and rapid response”quickly turn on and off entire pathways based on environment”

What is the purpose of 5’ caps?

Primary function of the 5’ cap is to protect the mRNA from degration by exonucleases

Recall that RNA can act as a ribozyme since it has catalytic functional groups and can adopt

specific 3D structures.

Recall that RNA can act as a ribozyme since it has catalytic functional groups and can adopt

specific 3D structures.

Note that some ribozymes are pure RNA, while others have protein

components (that are not involved in catalysis). Some ribozymes involve self-cleavage reactions

and thus cannot do multiple turnovers.

Note that some ribozymes are pure RNA, while others have protein

components (that are not involved in catalysis). Some ribozymes involve self-cleavage reactions

and thus cannot do multiple turnovers.

What causes RNAPII to escape the core promoter?

Phosphorylation of the CTD by TFIIH with mediator coordination

How does CTD phosphorylation allow elongation?

RNAPII dissociates from mediator and transcription factors and begins synthesizing full length mRNA

What is the purpose of 3’ poly (A) tails?

Poly(A) binding proteins associate with this tail to protect the mRNA from degradation and help control the mRNA’s lifetime within the cell.

What is the purpose of splicing in eukaryotic transcriptional processing?

How does the cell know where to splice the

exons?

Splicing occurs at conserved sequences at the exon-intron

junction.

How is the 5′ end of eukaryotic mRNAs

protected from exonucleases?

Eukaryotic mRNAs are capped at the 5′end by linking a

guanosine residue to the emerging mRNA via a 5′-5′

triphosphate linkage.

How is the 3′ end of eukaryotic mRNAs

protected from exonucleases?

Eukaryotic mRNAs are

polyadenylated at the 3′ end.

poly(A) binding proteins protect

the mRNA from degradation and

help control the mRNA lifetime.

How does the cell remove introns?

Splicing occurs through two transesterification reactions

catalyzed by an RNA-protein complex called the spliceosome

or catalyzed by the RNA itself.

What is the advantage of splicing?

Most human structural genes undergo alternative splicing

where different processing of the transcript produces variant

mature mRNAs.

How can RNA act as an enzyme?

RNA has multiple functional groups and adopts unique 3-

dimensional conformations just like a protein enzyme.

What is the mechanism of splicing?

The Mechanism of Splicing

Once these sites are identified, the spliceosome (a large complex of RNA and protein) or the RNA itself catalyzes two successive transesterification reactions to remove the intron and unite the exons:

1. First Transesterification: The 2' OH group of the branch point adenosine residue attacks the phosphate group at the 5' end of the intron. This reaction frees the 3' end of the first exon and creates a distinctive lariat-shaped intermediate (a loop of intron RNA).

2. Second Transesterification: The newly freed 3' OH group of the first exon then attacks the 5' phosphate of the second exon. This reaction forms a permanent phosphodiester bond that joins the two exons together, while the excised intron lariat is released and subsequently degraded.