RNA transcription

Molecular definition of a gene

Entire nucleic acid sequence that is necessary for synthesis of proteins OR rna.

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Genes are segments of DNA that are transcribed into RNA

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1. Resulting RNA encodes a protein (e.g., mRNA)
2. The resulting RNA functions as RNA and ay not be translated into protein (e.g., tRNA and rRNA)

More RNA means…

More protein

Transcription is for…

Creating RNA

1st rule: RNA is made \[….\] and \[….\] to DNA

Parallel, complementary

2nd rule: RNA is made in the \[..\] direction

5’ → 3’

The DNA template is read… direction

3’ → 5’

Sugar differences

Ribose has an OH group, deoxyribose does not

Is Uracil enough to determine whether something is RNA?

No, you can have dimers. you need to look for sugar differences.

Difference between Uracil and Thymine

Uracil is missing a Methyl group

An important difference between RNA and DNA

Ribonucleoside triphosphates used (ATP, UTP, CTP, GTP)

RNA Pol. Holoenzyme

Sigma factor and core enzyme

Transcription cycle (bacterial)

Steps:

Sigma factor binds to RNAP and finds promoter sequence

* Holoenzyme localized unwinding of DNA, abortive transcription (a few short RNAs synthesized initially) and RNAP clamps down, sigma factor released
* elongation (processive) phase
* Termination & release of RNA
* Bacteria have different sigma factors

Does RNA polymerase require a primer?

No

Is RNA polymerase accurate? Why?

No, because it has exonuclease activity (the backspace)

How is the promoter sequence numbered?

Upstream is the more negative number, downstream is more positive.

Where is the bacterial promoter

TTGACA to TATA box

Why does the sigma bean bound to the green sequences?

So that the active site is positioned over +1, right after the promoter consensus sequences

Where are the promoter consensus sequences shown and what are they?

The green sequences

* most common/average sequences
* -10, -35

Significance of the promoter

Positions RNA polymerase and determines whicg strand is template strand (3’ → 5’)

Hairpin loops

Formed due to base pairing with itself when the RNA transcript leaves RNAP

Terminator sequences

G’s and C’s followed b y A’s and T’s.

When the G’s and C’s bond, since they have 3 bonds they’re much stronger and form the hairpin formation. The DNA has weaker A’s, U’s, and T’s but since the hairpin is so strong it pries itself off of it

Key points about the transcription cycle

1) initial steps of RNA synthesis are relatively inefficient

2) elongation mode of RNAP is highly processive (moves very quickly)

What are some characteristics of RNA termination signals?

* Hairpin structure formed as a result of GC rich sequences
* AT rich DNA sequences following hairpin sequences (weak A-U bonding in the DNA/RNA duplex)

How do termination signals help to dissociate the RNA transcript from the polymerase

Disrupts H-bonding of new mRNA transcript with DNA template

@@Difference between sigma factor mode and elongation mode@@

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Key difference between prokaryotic gene expression and eukaryotic gene expression

Translation can begin before Transcription is complete because of the absence of a nucleus.

Eukaryotes have introns and exons, creating pre-mRNA transcript before translation begins.

5’ capping, RNA splicing and 3’ polyadenylation removes introns before mRNA leaves nucleus for translation.

Do exons have to be coding?

No, because EVERYTHING left over after transcription is exons, (5’ UTR and 3’ UTR) and that includes noncoding sequence. They are essential or else ribosomes do not work.

UTR stands for UNTRANSLATED (i.e., non-coding)

RNA types present in both Eukaryotes and Prokaryotes

mRNA rRNA tRNA

Do all RNAs help create proteins?

No

RNAP I

transcribes most RNA genes

RNA II

transcribes all protein-coding genes

RNA Pol III

transcribes tRNA genes

What are protein-coding genes

mRNAs

Eukaryotic RNAP II vs. Bacterial RNAP structure

Bacterial RNAP has 5 subunits, eukaryotic RNA Pol II has 12

* RNA Pol II has a %%SPECIAL carboxyl terminal domain (CTD)%% not found in bacterial or other eukaryotic RNAPs

Eukaryotic vs. bacterial RNA polymerases

Eukaryotic RNAPs require transcription factors

and also need to deal with chromosomal structures, like histones

Transcription factors

* proteins to help position RNAP at the promoter
* *similar* to the sigma subunit to bacterial RNA polymerases

Sigma subunit

bean-shaped protein behind active site of BACTERIAL RNAP

Eukaryotic promoters

* more variable than bacterial promoters
* 1 or more specific sequences called elements
* specific elements are at specific locations
* elements are recognized by specific general transcription factors, which in turn, help position the RNAP relative to the transcription start site

Elements

TATA box

sequence TATA is highly (evolutioarily) conserved, found \~30 bp upstream from start site for transcription (-30)

* Helps position RNAP II ANDDD general transcriptiion factors
* TATA box common, but many other types of promoter sequences (elements)

Steps in the initiation of transcription

Binding of TBP (TATA Binding Protein) subunit of TFIID (Transcription Factor (RNA pol II)) near TATA box sequences

* mobilizes the binding of TFIIB complex adjacent to TATA box
* Other transcription factors (TFs) bind
* RNAP II and other TFs will be able to bind in correct orientation at transcription start site
* SPECIAL: Helicase activity & Phosphorylation (Kinase activity) of C-Terminal Domain (CTD) of RNAP II -- both jobs performed by TFIIH
* Helicase activity separates strands, RNAP II is abortive until phosphyrlation happens, then it keeps on going
* OTHER RNAPs DO NOT HAVE THE CTD TAIL

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4 steps summarized:

1) TBP, a subunit of TFIID, binds to the TATA box promoter in the minor groove, bending and distorting DNA

2) this attracts other TPs, which help to orient and bind RNAP II to the DNA

3) the helicase activity of TFIIH uses ATP to pry apart DNA strands at transcription start site

4) TFIIH ALSO phosphorylates the C-terminal domain of RNAP II, activating it so that transcription can begin

Activator protein

binds to enhancer DNA sequences, folds over and kisses mediator, telling RNAP to go faster, getting out of abortive phase

RNAP II c-terminal domain

the carboxyl terminal domain of largest subunit has a stretch of 7 amino acids that is repeated multiple times (tandem repeats)

1 reapt = tyr-ser-pro-thr-ser-pro-ser

yeast enzyme has 26 repeats

human enzyme has 52 repeats

C-TERMINAL DOMAIN IS ESSENTIAL FOR VARIABILITY

How is RNAP II activated?

Phosphorylation

What is phosphorylated in RNAP II acitvation?

adding phosphate groups on S (Ser) located on the CTD

How many proteins are involved in initiating eukaryotic transcription?

>100 subunits of many proteins

mRNA Processing: Phosphorylation of C-terminal tail of _____ results in…

RNAP II

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binding of RNA processing proteins

Additional phosphorylation of CTD and phosphorylation pattern changes

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YOU RARELY GET THE FULL UNPROCESSED PRE-MRNA

5’ pre-mRNA capping

Helps to protect RNA from 5’ → 3’ exonucleases

completed BEFORE mRNA fully transcribed

Removal of introns from pre-mrna

1) branch-point A attacks the 5’ (OH) splice site

2) 3’ of on exon reacts with 5’ of next exon to release intron

THIS ALL HAPPENS THROUGH A SPLICESOME COMPLEX

Catalytic mechanism is RNA dependent (significance of what functional group?)

3) 2’OH group of the ribose sugar is not present in deoxyribose

4) this group is necessary for the formation of the lariat structure in intron splicing

snRNPs

pre-mRNAs not able to self-splice

Splicesosomes contain snRNAs bound to protein -- (snRNPs) plus other associated proteins

Spliceosomes assemble on mRNA to remove introns

When splicing complete, exon junction complex added to signify completion of splicing

Correct sequences required for accurate mRNA splicing

Why does alternative RNA splicing increases the number of gene products?

Because different kinds of splicing results in different kinds of gene expression (different kinds of mRNA)

Abnormal splicing

You can have exon skipping, cryptic (incomplete) splice sites, or new splice sites created causing new exons to be incorporated

Termination in eukaryotes

3’-end modifying proteins

Gu-rich or U-rich sequences are encoded in the genome

but after they are transcribed, the 3’ end processing proteins recognize them on the mRNA and are recruited to the mRNA

note that the poly-A sequence is NOT encoded in the genome

mRNA processing (CTD phosphorylation)

CTD phosphorylation happens because, each time, it allows a different protein to bind to the tail.

change in phosphorylation happens each time for:


1. move capping proteins from CTD to 5’ end of growing mRNA from RNAP
2. move spliceosome over to splice
3. move 3’ end processing proteins

3’ end processing

CPSF (Cleavage (meaning cuts RNA) and Polyadenylation specificity factor) and CstF (Cleavage Stimulation Factor)

poly-A signals (Ts) transcribed to AAUAAA

CPSF and CstF transferred over to RNA

CPSF cleaves RNA CPSF is still binded

poly-A-polymerase (PAP) directly adds (binds) a bunch of A sequences to RNA after AAUAAA, using ATP. CPSF is still binded.

Length is regulated when PAP terminates, poly-A-binding proteins protect 3’ end from 3’ → 5’ exonucleases on mature 3’ end of an mRNA molecule

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in summary:

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→ consensus sequences (yellow portion in picture) direct cleavage and polyadenlyation of the 3’ end.

→ 3’ end processing proteins move from CTD to MRNA

→ Cleavage and addition of a poly-A 3’ tail along with Poly A-binding proteins result in the mature mRNA

mRNA export

* exported from nucleus to cytosol
* cap-binded protein binds to 5’ end
* exon junction complex also binds to where introns used to be
* note: they are not binding the exons, they’re just.. there
* poly-A-binding protein and sequence at 3’ end
* exits through nuclear pore complex
* In cytosol, cap binding complexes can be removed, and initiation factors for protein synthesis bind, leading to translation