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3 steps that convert premrna to mature mrna

5’ capping

rna splicing

polyadenylation

rna splicing utilizes the __ to remove ? and join ?together

Spliceosome

introns

extrons

Introns removed as a loop are called a

lariat

spliceosome contains what type of rna

snRNA

what is branch point adenine

specific adenine nucleotide inside an intron

Describe rna splicing with branch point adenine

Specific nucleotide (A) on intron uses 2OH group to attack 5’ of same intron

5’ site on intron binds to 2oh group, forming a lariat

3’OH group on nearby Extron reacts with 3’ site on intron, separating from other Extron

exon 1 joins exon 2 through phosphodiester bond

Polydenylation refers to

addition of poly-tail to 3’ end of preMRNA

AAUAAA refers to

polyadenylation signal sequence

what does the polyadenylation signal sequence do

recruit protein

signal cell where to cut

The cleavage site that occurs from polyadenylation is usually __ nucleotides from the Poly A signal

30

What major protein complex binds to AAUAA sequence + function

CPSF

position cleavage site

What binds to a GU rich region downstream a cleavage site + function

CstF

position cleavage site

After cleavage, new 3’ recieves new adenine nucleotide from what polymerase

pol a

Does Pol A need a template

no (fuh no)

Polyadenylation is important as it

protects mRNA from degradation

helps export mature mRNA

What is 5’ capping briefly

adding modified guanine nucleotide (7-methylguanosine) to 5’ end of preMRNA

What does 5’ capping achieve

protection of mRNA from degradation

Which enzyme forms the unusual 5′–5′ linkage during mRNA capping?

Guanylyltransferase

Describe 5’ capping in detail

RNA triphosphatase removes a phosphate from newly synthesized 5’ rna

Gunalyltransferase adds G nucleotide from GTP

Methyltransferase adds methyl group

What does the unusual 5′–5′ linkage achieve

protection from mRNA enzymes that degrade from 5’ end

pre-mRNA (heterogenousnuclear RNA) refers to

First rna transcribed from DNA (primary transcript)

snRNA means what and functions in what

Small Nuclear RNA

Forms splicing mechanism that processes preMRNA to mRNA

allows splicing of non coding (intron) sequences out to allow extrons to join

Transcription vs translation quick lil memory trick (which occurs first

transcription occurs first because translation is LATE

__ is a non protein enzyme

snRNA

tRNA means what and functions in what

transfer RNA

carries amino acids to ribosome during translation

rRNA means and functions in

ribosomal RNA

combines with proteins to form ribosome

catalyze peptide bond formation between amino acid during translation

Which of the three preMRNA processing events can be tracked with electrophoresis

rna splicing (maybe polyadenylation too)

How can electrophoresis be used to examine RNA splicing

RNA splicing removes introns and shortens RNA, this change can be examined

what is required for pre-mRNA splicing and why

5’ splice site=identify beginning of intron

BranchPointAdenine=2OH group initiates lariat formation

3’SpliceSite=end of intron

SpliceosomeAndATP=Support splicing process

What is required for Polyadenylation and why

AAUAAA=Signal for CPSF recognition

Downstream GU sequence=Signal for CstF recognition

Cleavage Factor=Cut premRNA to make new 3’ end

Pol A and ATP= add adenine nucleotide

What is required for 5’ capping

5’ triphosphate on new RNA=provides end to be capped

Capping enzymes=remove phosphate, add guanine, add methyl

GTP/SAM=provide guanine + methyl

Svedberg units for prokaryotes

30/50 = 70

Svedberg units for eukaryotes

40/60 = 80

Initiation process of translation in detail

Small subunit joins 5’ end of mRNA transcript

Scan for start unit (AUG)

initiator tRNA pairs its UAC anticodon with AUG

Large ribosomal unit join

(INITIATION COMPLEX IS FORMED THROUGH ALL THESE)

Elongation process of translation in detail

Charged tRNA enters A site and pairs its anticodon with mRNA codon

rRNA catalyzes reaction that links protein in P site to A site

Ribosome moves forward one codon, shifting the empty tRNA from P to E and growing protein from A to P

Empty tRNA exits

tRNA follows what direction in respects to ribosomal locations

APE

Aminoacyl site describes the

arrival site where

tRNA carries an amino acid

anticodon pairs with complementary mRNA codon

delivers next amino acid needed for protein

Anticodons refer to

three nucleotides on tRNA that pairs with complementary codon on mRNA during translation

What is aminoacyl-tRNA synthetase + function

enzyme that uses ATP to attach correct amino acid to corresponding tRNA

Wobble pairing refers to ?

What is achieved?

flexible pairing at 3rd nucleotide of mRNA and 1st nucleotide of tRNA

allows one tRNA to recognize multiple codons for same amino acid

Template DNA strand is complementary to new

mRNA strand

Coding DNA strand is identical to __ strand, except it uses U instead of T

mRNA strand

What helps initiate eukrayotic translation

5’cap

Both 5’ cap and poly-A tail achieve

protection of mRNA from degradation

Relationship of # of AUG sites and # of proteins?

Not every AUG codon starts a new protein, allowing multiple AUG codons within one protein coding sequence

What is a polysome? Advantages?

group of multiple ribosomes translating same mRNA molecule

increased protein production / efficiency

molecular chaperones, what are they what do they do

proteins that help other proteins fold properly and prevent incorrect folding or aggregation

Hsp70

molecular chaperone that binds exposed regions of unfolded proteins and helps prevent misfolding and aggregation

What is aggregation

when multiple proteins stick together and form clumps

what is ubiquitin

small protein used as a marker for other proteins to be degraded

Proteasome

protein complex that recognizes and breaks down ubiquitin tagged proteins into peptides

Which enzymes attach ubiquitin

e1

e2

e3

e1 enzyme role in ubiquitination

activate ubiquitin using ATP

e2 enzyme role in ubiquitination

carry activated ubiquitin

e3 enzyme role in ubiquitination

recognize target protein and help transfer ubiquitin onto it

26s proteasome role

degrade ubiqutin tagged proteins

Stop codons

UAA (U ARE ANNOYING)

UGA (U GO AWAY)

UAG (U ARE GAY)

In what direction does RNA replication read

3-5

In what direction does RNA replication produce

5-3

mRNA that is newly synthesized is ___ to the DNA template

complementary

mRNA that is newly synthesized is __ to the DNA coding strand

identical, with the exception of U instead of T

In what direction does the ribosome read mRNA

5-3

In what direction does the ribosome produce protein

N-C

What does Northern Blot reveal?

quantification of specific RNA

Explain in detail how Northern Blot works

Isolate RNA in agarose gel via electrophoresis

Transfer RNA to membrane favored by membrane positive and RNA negative charges

Label RNA with probe complementary to RNA

Dark band=more RNA

What direction tests reveal what?

Northern RNA

Southern DNA

Western Protein

What does NPA reveal

which regions of DNA are protected by nucleosomes (DNA wrapped in histones)

Explain the process of NPA in detail

Enzyme cuts DNA not protected by nucleosomes

Protected DNA remains revealing nucleosome organization and chromatin accessibility

An NPA where DNA is tightly bound reveals what about transcription

little/no transcription

In an NPA, what happens to accessible DNA regions

Nuclease digestion cuts as its not protected by nucleosomes

Unexpressed genes in an NPA are tightly wrapped around histones, which

protects DNA from degradation in the assay

Relationship between Northern and NPA

Northern reveals how much RNA whereas NPA reveals how much of the DNA is protected

Used together to reveal how DNA packaging explains difference in RNA

Transcription rate refers to

how frequently a gene is transcribed into RNA

Explain in detail why transcription rates differ

Transcription Factors like activators and repressors can increase/decrease

Promoter strength can allow recruitment of more transcription machinery

Chromatin structure allows euchromatin to be more accessble

In respects to degradation, how can different levels of protein be made in transcription

Higher protein degradation yields low protein abundance

Name the enzymes involved in RNA/DNA synthesis

RNA Pol

DNA Pol

Name the nucleotides involved in RNA synthesis

ATP UTP GTP CTP

Name the nucleotides involved in DNA synthesis

dATP dTTP dGTP dCTP

Name the bases of RNA

AUGC

Name the bases of DNA

ATGC

Is a primer required for rna synthesis

no

RNA pol1 makes

Ribosomal RNA (rRNA)

RNA pol2 makes

mRNA

miRNA

snRNA

RNA pol3 makes

tRNA

5s rRNA

Upstream regulator proteins for transcription

activator

repressor

Describe in detail how initiation complex forms in transcription

TFIID binds promoter using its TBP to recognize promoter (TATA box)

TFIIA TFIIB stabilize complex and position RNA Pol 2

RNA pol2 and TFIIF join

TFIIE and TFIIH join

TFIIH opens DNA and phosphorylates CTD on RNA Pol 2

upstream regulatory dna sequences for transcription

enhancer

silencers

Sense strand refers to … functions in…

portion of loop not involved in RNA Pol moving along

Same sequence as RNA except T goes U

what TF opens transcription bubble

TFIIH

describe in detail the process of elongation in transcription

RNA pol moves along DNA template 3-5

DNA unwinds ahead

Complementary RNA nucleotides added to 3 of growing RNA STRAND

phosphodiester bonds between RNA Nucleotides

DNA reanneals behind RNA pol

Prokaryotic vs Eukaryotic, the promoter is recognized by

sigma factor

TFIID containing TBP

What are the sequences of -35 and -10 AND their functions

TTGACA, sigma factor recognition

TATAAT, transcription initiation

-35 -10 are unique to

prokaryotes

sigma factor combines with RNA pol to form a

holoenzyme

Holoenzyme recognizes promoter on DNA and binds to it, causing

local unwinding of DNA near -10 (transcription bubble)

After DNA is locally unwound in prokaryotes, ___ must occur. _ Is the process of

Abortive initiation

repeatedly synthesizing and releasing short RNA transcripts before successful escape of promoter

Abortive initiation is important as it

releases short unstable RNA, allowing stable RNA to overcome promoter interactions, starting elongation

What is the threshold for successful initiation

10 nucleotides

How do you determine which strand is used as a template

identify strand that runs 3-5 in direction of rna pol

What transcribes all protein coding genes

rna pol2