Post Transcriptional Regulation

Post transcriptional regulation takes place when?

after transcription, but before translation

after DOES NOT mean that transcription has to be complete

can occur while transcript is still being transcribed

Structural and spatial differences in gene expression between prokaryotes and eukaryotes

eukaryotes have a nucleus (where transcription occurs), mRNA is exported to cytoplasm after post transcriptional regulation for translation

prokaryotes do not have regulation after transcription and translation

___________ intervenes between exons

introns

Exons

protein coding regions

only about 1.5% of the human genoms

Introns

noncoding region

over 23% of the genome

Most introns are ________________, they need to be removed via _________________

untranslatable, splicing

Splicing

introns are removed and exons are joined together

Where does post-transcriptional regulation occur?

nucleus

Nuclear mRNA processing involves:

capping and methylation

splicing

polyadenylylation

What happens to primary transcripts (pre-mRNAs) as soon as they are transcribed by RNA pol II?

they are capped

What caps pre-mRNAs?

guanylyl transferase using GTP as a substrate

The capped G residue is _______________ at _______________ position

methylated, N7

What happens at the next two residues beside the G residue at position N7?

additional methylation occurs at C2'-O positions at the next two residues and at 6-amino group of the first adenine

A cap with only a single -CH3 on the guanyl is termed

cap 0

What does cap 0 occur in?

all eukaryotic mRNA

If a methyl is also added to the 2'-O position of the first nucleoside after the cap, what structure is generated?

cap 1

(predominant cap form in all multicellular eukaryotes)

In the capping of eukaryotic pre-mRNAs, guanylyl transferase catalyzes the addition of _________________ derived from ______________ to the 5' end of the growing transcript. What is liberated?

guanylyl residue, GTP, PPi is liberated from GTP and the terminal phosphate is removed from the primary transcript

What is done to the third phosphate group on the 5' end of the primary transcript?

a phosphatase removes it

Guanylyl transferase next brings in ____________, which goes on the ____________ end. Once ______________ is added, _____________ is eliminated

GTP, 5', GTP, pyrophosphate (PPi)

What adds a methyl group to the guanine on the 5' end?

guanine 7-methyltransferase

The 5' cap is recognized by

cap binding proteins

The 5' cap distinguishes mRNA from other types of RNA molecules such as

RNA molecules produced from RNA pol I and pol III. They each produce uncapped RNAs

What RNA pols produce capped RNAs?

only RNA pol II --> mRNA

What do mRNAs have to have for export from the nucleus?

a cap and a poly A tail

Is a 5' cap necessary for translation?

yes

What does the 5' cap of mRNA do for the mRNA in the cytoplasm?

stabilizes it and prevents it from being degraded

When does termination of transcription occur?

only after RNA pol has transcribed a concensus AAUAAA sequence (polyA signal)

What is the polyA signal?

AAUAAA

10-30 nucleotides after the polyA signal, what happens to the mRNA?

it is cleaved and a string of ~200 adenine residues is added to the mRNA transcript --> polyA tail

Enzyme that adds the adenine residues to produce a polyA tail

polyA polymerase

polyA tail bound by _____________ stimulates translation and governs the stability of mRNA

PABP

polyA binding proteins

Where does polyA addition occur?

3' end of transcripts about 10-35 nucleotides downstream from the polyA signal (AAUAAA)

What binds to the polyA signal and mediates looping of the 3' end of the transcript through interactions with a G/U rich sequence even further downstream?

CPSF

cleavage and polyA specificity factor

What do cleavage factors do?

bind and bring about the endonucleolytic cleavage of the transcript to create a new 3' end 10-35 nucleotides down from the polyA signal

Does polA addition require a template?

no

Is the polyA tail encoded in the genome?

no

it is added by polyA polymerases

What is the function of the polyA tail?

by interacting with PABP, it is necessary for efficient translation and protection from mRNA degradation

First amino acid of all proteins

methionine

AUG

In the nucleus, pre-mRNA forms _____________ by associating with a characteristic set of nuclear proteins

ribonucleoprotein particles (RNPs)

What do the nuclear proteins help maintain pre-mRNA as?

untangled and accessible

Where does splicing occur?

nucleus

Substrate for splicing

capped, polyadenylated RNA in the form of a RNP complex

What happens in splicing?

the introns are excised and the exons are sewn together to form mature mRNA

What is the 5' end of an intron in higher eukaryotes?

GU

What is the 3' end of an intron in higher eukaryotes?

AG

What is present about 18-40 nucleotides upstream of introns' 3'?

branch site

The branch site is essential to what?

splicing

Branch site is usually

YNYRAY

Y= pyrimidine

R=purine

N=anything

A covalently closed loop of RNA is formed by attachment of the _________ of the intron's invariant 5'-G to the 2'-OH at the branch A site

5'-P

Splicing depends on what ribonucleoprotein particles?

snRNPs

A snRNP consists of

small nuclear RNA and about 10 different proteins

Spliceosome

snRNPs and pre-mRNA

Lariat intermediate

the 2' OH of the branch point adenine attacks the donor sequence, cleaving the 5' splice site

Splicing occurs when what come together?

various snRNPs come together with the pre-mRNA to form the spliceosome

What does the assembly of the spliceosome require?

ATP

snRNP U1 binds to

5' splice site

snRNP U2 binds to

branch site

Interaction between U1 and U2 snRNP does what?

brings the 5' and 3' splice sites together so lariat can form and exon ligation can occur

5 different snRNAs

U1, U2, U4, U5, U6

The 5 snRNAs are rich in

uridine

What is associated with each of the snRNAs to form snRNPs?

proteins

U1 snRNP binds at the ____________, followed by the association of U2 snRNP with ________________

5'-splice site, UACUAA branch point sequence

The _____________ replaces U1 at the 5' splice site and directs the __________ of the branch point sequence with the 5' splice site, where ________________ is released

triple U4/U6-U5 snRNP complex, juxtaposition, U4

Constitutive splicing

every intron is removed and every exon is kept in the mature mRNA

produces a single form of mature mRNA from the primary transcript

How might eukaryotic genes give rise to multiple forms of mature RNA transcripts?

using different promoters

selecting different polyA sites

alternative splicing of the primary transcript

OR A COMBO OF ALL 3

Alternative mRNA splicing

different cell types express distinct isoform

Alternative splicing expands the

coding potential of the genome

Are bacterial mRNAs unstable or stable?

unstable

Bacteria can/cannot adapt quickly to environmental changes?

can

Most eukaryotic mRNAs are more/less stable than prokaryotic mRNAs?

more

What removes the polyA tail?

polyA nuclease

mRNA is thought to be circularized by its interaction with

eIF4E, eIF4G, and PABP

PABP, EIF4E and EIF4G circularizing the mRNA does what for the mRNA?

protects the 5' and 3' ends from attack by decay enzymes and stimulates translation

RNA editing

process that changes one or more nucleotides in an RNA transcript by deaminating a base

either A-->I or C--> U

A to I RNA editing is carried out by

ADAR

adenosine deaminase acting on RNA

General decay pathway of most mRNAs

Slow polyA shortening

decapping

5'-->3' degradation