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Flashcards in RNA Structure And Synthesis Deck (38):
1

Amount of RNA in cell vs DNA

40x more RNA than DNA in the cell

2

DNA vs RNA

DNA
-master copy
-carefully reproduced
-stable
-nucleus

RNA
-working copy
-so tangly synthesized, used and degraded
-cytoplasm

3

Which is larger: eukaryotic cell or bacterial cell?

Eukaryotic cell

4

Which is larger: mRNA or primary transcript?

Primary transcript

5

Similarities between DNA and RNA structures

-long unbranched polymers of nucleotides linked by 3'-5' phosphodiester bonds

-sugar phosphate backbone

-3 common bases: adenine, guanine, cytosine

6

Difference in DNA and RNA structures

-sugar in RNA is ribose
-RNA contains uracil instead of thymine
-RNA is shorter than DNA
-always single stranded
-RNA can form secondary loop and stem structures (i.e. tRNA)

7

Sort types of RNA from most abundant to least

rRNA --> tRNA --> mRNA

8

Basics of Transcription

Sequence dependent

Anti-parallel direction for DNA and RNA
-DNA read 3' --> 5'
-RNA synthesized 5' --> 3'

Asymmetrical: only one strand is transcribed

Does not require a primer

9

Coding (sense) strand

the DNA strand that goes 5' --> 3'

The sequence that correlates with the mRNA

10

Template (non-coding) strand

DNA strand that goes 3' --> 5'

Strand that is copied during RNA synthesis

11

Beta subunit on RNA polymerase

Contains the catalytic site- catalyzes 3' --> 5' phosphodiester bonds

12

Alpha subunit on bacterial RNA polymerase

Recognizes promoter regions, which is required for appropriate initiation of transcription

13

Does RNA polymerase transcribe the entire genome?

Nope

14

Subunits of the core enzyme

Alpha (2) (interacts with proteins)
Beta
Beta' (binds DNA template)

15

Subunits of the holoenzyme

Same as core + sigma

16

Is transcription discontinuous or continuous?

It's discontinuous --> different start/stop signals

Either strand can be a coding template

17

Prokaryotic Promoter Region

Upstream of the start site of transcription initiation (++1)

Written on the sense strand

Specifically recognized by sigma subunit

Pribnow sequence and -35 region : signals RNA polymerase to come and start transcription

18

What is important for efficient initiation of transcription?

The distance between pribnow box and the -35 region

Not every gene has the same Pribnow and -35 sequence

19

Transcription: Initiation

Sigma binds to specific promoter regions of DNA (-35 and -10)

These regions correspond with DNA sequence...if there was a different sequence in between, sigma would not bind

20

Steps in transcription initiation

1) RNA pol binds to DNA- RNA pol for enzyme is always bound to DNA in a non-specific manner

2) when sigma subunit binds to core enzyme at promoter site, a conformational change occurs and binding is stabilized (closed promoter complex)

3) After formation of the closed promoter complex, the 2 strands of the DNA template unwind to produce and unpaired region containing approximately 12 bases (open promoter complex)

4) 1st base in RNA chain is added complementary to the base at +1 of the template strand

5) After 8-10 bases are added, the sigma subunit leaves the complex and only the core enzyme remains

21

Role of beta subunit of RNA polymerase in initiation

Beta subunit has 2 nucleotide binding sites
1) for 5' purine (therefore first base is a 5' purine)
2) for subsequent NTPs

Also synthesized the phosphodiester 3'--5' bond between NTPs

22

Transcription Elongation

RNA-DNA hybrid about 8 bases long

Transcription bubble always 12 bases

As the enzyme moves to the region in front, creates + super coils and the region behind it re-winds creating negative supercoils

23

Hairpin formation in transcription termination

Must be a GC rich palindrome (very stable)
Must be followed by UUUs

24

Rho-independent termination of transcription

Pol slows down at GC rich region

Hairpin forms

Weak A/U bonds

25

How does the inverted repeat of rho independent termination end?

Stops at T-A spots

26

What is Rho

Rho is a large hexameric protein-Helicase, ATPase

Works in the 5' --> 3' direction

27

Rho-dependent Termination of Transcription

Rho binds to the RNA transcript

Polymerase slows at a C-rich pre-termination sequence

Rho moves to the 3' end of the RNA transcript, hydrolyzing ATP

Uses Helicase activity to unwind DNA-RNA hybrid--> to release RNA from complex

Dissociation of Rho and RNA polymerase from the RNA

28

Key differences between transcription in eukaryotic vs prokaryotic cells

Eukaryotes have:

3 polymerase so
No sigma subunit
Polymerase-protein interactions, not pol-DNA interactions govern transcription initiation

29

RNA polymerase II

Has no sigma factor to locate promoter region but interacts with specific proteins which bind core promoter regulatory elements

Transcribes the precursors of mRNAs (primary transcript)

Enables specific genes to be expressed in different developmental stages, different tissues, and in response to different environmental stimuli

30

RNA Pol LL Promoter/Enhancer Regions

More complicated than prokaryotes --> there aren't any promoter sequences or organization like that

Enhancer- regulates efficiency of transcription

Constitutive- will be specific to the tissue its in

Basal- where it starts

TATA box- present but not in all genes (determines where transcription starts)

*everything depends on tissue and what sort of gene is being transcribed*

31

Basal Transcription Complex- Eukaryotes

TFII- transcription factors for Pol II

TBP- TATA binding protein

TAF- TATA associated factors

Protein-protein interactions are key to initiation
Formation of open promoter complex involves ATP hydrolysis
TFIIH= kinase which phosphorylates Pol II

Transcription initiation

32

Transcription elongation- basal apparatus

Elongation phase similar to that in prokaryotes

Dephosphorylation involved in termination

33

Inducible region

Specificity of Tx

Hormone receptor binding

34

Enhancer Region

Activator binds

35

CAAT Boc

Stabilized binding of pol 2

Controls of Tx (along with GC box)

36

What allows for efficient transcription?

The 3D structure of protein and polymerase II and the gene

Chromatin remodeling, histone acetylation/deacetylation also involved in efficiency of transcription initiation

37

Use of Tamoxifen

In order to identify potential gene targets, an estrogen receptor positive breast cancer cell line grown in culture is given tamoxifen

RNA is slanted from untreated cells and cells treated with different doses of tamoxifen and microarray analysis is performed to identify upregulated (red) and down regulated (green) genes

The upregulated genes are likely regulated by binding of the ER tamoxifen complex to an Inducible element (i.e.: tamoxifen)

38

Alpha- amanitin

Inhibits RNA Pol II >>> Pol III not Pol I

No mRNA = no protein synthesis = liver failure, death