Transciption Flashcards by Jeremy Penn

Action of RNA Polymerase

How does it begin?
How does it read the template?
How does it synthesize the transcript?
What does it recognize?

Begins transcription WITHOUT a primer

Reads the template strand from 3’ to 5’

Synthesizes RNA transcript 5’ to 3’ using ribonucleotides

Recognizes transcription termination signals

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What is an NTP?

Ribonucleotide

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Transcription begins with..

The RNA polymerase identifies a genes’ PROMOTER region

Interacts with the DNA template strand

Initiates synthesis of a complementary , anti parallel RNA transcript

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Transcription ends when…

A termination signal is reached

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What is described by the sequence of the coding strand?

Genes.

However, the template strand is actually the strand of DNA read during synthesis.

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The DNA coding sequence is identical to:

The RNA transcript sequence, instead of T’s it uses U’s

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How are base sequences labeled (downstream to the right)?

And upstream DNA bases (to the left)?

+1, being the first base transcribed, then +2 +3, etc in the 3’ direction

Upstream -1, -2, -3, etc in the 5’ direction

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Using what method, how do you determine the other two strands being transcribed/translated?

Watson-Crick base pairing

ATGGGG -> TACCCC -> AUGGGG -> Met Gly

DNA -> transcribed strand -> translated strand -> protein

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What type of molecule is a Prokaryotic RNA polymerase?

Holoenzyme

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What makes up the holoenzyme?

Core enzyme + sigma factor

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What makes up the core enzyme of the holoenzyme?

4 subunits.

2 alpha subunits
1 beta subunit
1 beta-prime subunit

α2ββ’

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The importance of the core enzyme and sigma factor parts of the holozenzyme/RNA polymerase

Core enzyme- has RNA polymerase activity

Sigma factor- is required for recognizing and binding promoter sequences

Looks like an upside down chef’s hat. The base is the sigma factor, the top is the core enzyme

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Role of the sigma factor (of the holoenzyme)

Different sigma factors recognize different types of promoter sequences

They recruit the core enzyme to the DNA promoter (since they’re attached)

Dissociates from the core enzyme after transcription begins

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Initiation of transcription

Scanning and recognition of promoter sequence cues by the holoenzyme trigger transcription initiation.

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What do the promoters contain?

Contains two consensus sequences that are recognized by sigma factor.

TATA box
-35 sequence

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TATA box

Part of the promoter region

Consensus sequence of 6 nucleotides

TATAAT

Aka Pribnow box, ~7 nucleotides upstream from the +1 transcriptional start site (~-7)

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Another name for the TATA box

Pribnow box

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What is the -35 sequence?

Part of the promoter region

Consensus sequence of 6 nucleotides

TTGACA

~35 bases upstream from the +1 transcriptional start site

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What is elongation?

When transcription begins at the +1 transcriptional start site.

Sigma factor is released and the core enzyme continues

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The elongation process contains the core enzyme which does what?

Creates a temporary “melt” between the double stranded DNA

-forming a transcription “bubble”

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What can supercoils of the DNA strand be relieved by?

The action of topoisomerases

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Net reaction of Prokaryotic RNA Synthesis

Addition of a ribonucleotide to the growing RNA chain, and the release of pyrophosphate

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Pyrophosphate is further cleaved by:

Pyrophosphatase.

Irreversible coupled reaction.

2 high energy bonds are cleaved.

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What are the 2 termination mechanisms of Prokaryotic RNA synthesis?

Rho-dependent termination requires an additional protein.
(Rho factor: displaces the DNA template strand from RNA polymerase)

Rho-independent termination requires a G-C rich stem loop followed by a poly-U stretch
(Causes RNA polymerase to dissociate)

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Prokaryotic mRNAs can be polycistronic..what does that mean?

One mRNA can code for several proteins

Polycistronic mRNAs often do what?

Code for multiple enzymes in the same biosynthetic pathway

Eukaryotic mRNAs are always polycistronic. T/F

FALSE They're always MONOcistronic

What is rifampin?

Aka rifamycin. An antibiotic Binds to prokaryotic RNA polymerase (changes its confirmation) and prevents transcription initiation/elongation

What is rifampin/rifamycin used to treat?

Prokaryotic synthesis of tRNA and rRNA, how is it different from eukaryotic RNA synthesis?

Similar to model for Eukaryotes, except in prokaryotes the same RNA polymerase produces all types of RNA

Eukaryotic DNA is highly condensed into __________. But for gene transcription, the genes must be exposed. This "exposure" occurs in the ____________. Why?

Chromatin. Euchromatin, less condensed area, more accessible to RNA polymerases.

What causes the chromatin to revert to nucleosomes?

Acetylation of histone H1

Heterochromatin vs Euchromatin

Euchromatin- less condensed Heterochromatin - appears more dense, DNA is relatively inaccessible due to highly condensed structure

What is special about DNA in heterochromatin?

Its highly methylated

DNA methylation characteristics is the foundation of..

Epigenetics

Eukaryotic RNA polymerases

I, II, and III

Transcribes precursors to rRNA in the nucleolus

RNA polymerase I

Transcribes the precursor to mRNA (hnRNA) in the nucleoplasm (some snRNAs as well)

RNA polymerase II

Transcribes tRNA precursors (also small rRNA (5S) and some snRNAs)

RNA polymerase III

Transcription initiation in eukaryotes begins with what RNA polymerase?

RNA polymerase II

RNA polymerase II recognizes two consensus sequences which are... For transcription initiation in eukaryotes

TATA box (Hogness box) - similar to TATA box in prokaryotes - approximately 25 nucleotides upstream (-25) CAAT box -approximately 70 nucleotides upstream (-70)

What is different about elongation and termination in Eukaryotic RNA synthesis compared to prokaryotes?

Elongation is essentially the same. Termination in eukaryotes requires a signal...that isn't exactly understood. Hmm.

rRNA Synthesis and Processing

3 rRNAs are transcribed as a single large precursor (by rRNA pol I in the nucleolus) Individual rRNAs cleaved apart by RNases Prokaryotic rRNA synthesis is the same as in eukaryotes

What transcribes the three rRNAs from a ribosomal RNA gene? What cleaves the 3 RNAs from a chain to individual rRNAs?

RNA polymerase I RNases

Where is RNA pol III?

In the nucleoplasm

What is RNA pol III responsible for?

In tRNA synthesis and processing: Removal of intron loops. Trimming the 5' and 3' ends Base modifications Addition of the 3' -CCA sequence

Where does eukaryotic mRNA synthesis occur? Transcription and translation

Transcription - in the nucleus Translation - in the cytoplasm

Eukaryotic mRNAs vs Prokaryotic mRNAs

Euk mRNAs are extensively processed. -hnRNA to mRNA Pro mRNAs begin translation before transcription finished.

What are the MAJOR differences between Pro/Euk RNA synthesis?

Pro- promoter regions, 5' and 3' UTRs, coding region Euk- 3' terminus (and 5' terminus), exons and introns are present

What protects eukaryotic mRNA from nuclease degradation?

5' capping addition of 7-methylguanosine to the 5' end of the mRNA 5' to 5' linkage

What is the 3'-poly-A tail on euk mRNA? What does it do? What does it serve as? Is it encoded in the gene?

The 3' terminus contains a polyadenylation signal - Poly-A polymerase, a special enzyme that adds a variable stretch of adenine residues (~40-200) Serves as a signal for transport out of the nucleus, stabilizes the mRNA from nuclease attack. Not encoded in the gene. The SIGNAL sequence is encoded, but not the adenine additions.

Intervening sequences

Introns

Expressed sequences

Exons

What is the fate of introns?

Must be removed from the coding region for proper translation

The removal of introns and the joining of exons by snRNPs

Splicing

Composed of snRNAs and a group of proteins

snRNPs

snRNPs combined with the primary transcript The molecular machine that performs the splicing reaction

Spliceosome

What are splice sites?

Have very specific sequences, mutations at these sites can cause incorrect splicing

Exons are joined together during splicing to form..

Mature mRNA

What is a lariat?

The excised intron

Eukaryotes can contain how many intron sequences?

0-50

Certain hnRNAs may undergo alternative splicing. What is that? What's the point?

Joining of different exons together to form different mRNAs It permits the production of proteins with some common domains, but overall the function is different.

Systemic Lupus Erythematosus What is it? Who does it usually affect? Why is it a concern?

Autoimmune disease. Late-teen females Antibodies are produced that recognize host proteins, including snRNPs. Other pts can produce antibodies that recognize nucleic acids (anti-dsDNA) **anti-nuclear antibody panel is performed to counter this

Eukaryotic mRNA Processing summary

- processing events - 5' capping - 3' poly-adenylation - splicing - after all processing events are complete, mature mRNA is transported to the cytoplasm

Mushroom Poisoning What type of mushroom? What does it do?

Amanita phalloides, the death cap mushroom. OOOOoooOOoo Alpha-amantin binds to RNA polymerase II and inhibits mRNA synthesis. Accounts for 95% of all mushroom fatalities

Transciption Flashcards

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