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80 Cards in this Set

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Protein synthesis begins_____ to _____

amino terminus to carboxyl terminus

An mRNA with many attached ribosomes is called

polysome

5 stages of protein synthesis

Activation: charging of aminoacyl tRNA


Initiation(IF): assembly of mRNA, initiation tRNA, ribosome


Elongation: Fomration of peptide bond, translocation mRNA


Termination and release: water hydrolysis of peptide


Folding and sorting

3 tRNA binding sites on the 30s and 50s subunit of the ribosome

A site: aminoacyl site where incoming tRNA bind


P site: peptidyl site where the growing protein is held


E site: exit site where deacetylated tRNA is held

Important intiation factors for translation

fMet-tRNA: special initiator tRNA, distinguishes start AUG from internal methionine




IF2 (GTP) - brings in only fMet-tRNA




16s rRNA (30s)

Steps for translation initiation

16s rRNA positions AUG in mRNA into the P peptidyl site


IF2 (GTP) brings in fMet-tRNAf to AUG


fMEt tRNA anticodon pairs with AUG


Hydrolysis of GTP bound to IF2


Release of IF1, IF2(GDP) and IF3


50S subunit binds

How is AUG identified during translation intitation?

using the shine and delgarno sequence in mRNA


The SD sequence is present in the untranslated non coding portion of the RNA

What is the SD sequence

a purine rich element located about 10 nucleotides 5' of AUG

The SD sequence base pairs with _____

base pairs with 16s rRNA in the 30s subunit to position the AUG in the P site

______ is the initiator amino acid in bacteria

N formylmethionine

What does fMet-tRNA bind?

only the first initiating AUG codon and not AUGs elsewhere in the mRNA

What type of AA synthetase adds methionine to fMet tRNA

the same one that adds MET to tRNAs that add methionine to middle

What happens once methionine is bound to tRNAf?

A specific transformylase modfies the methionine once it is attached to tRNA, add formyl group

What are the steps of translation elongation

EF-Tu(GTP) brings in AA tRNA to A site


Codon-anticodon pairing induces GTP hydrolysis and EF-Tu(GDP) release, contributes to accuarcy


The PTC positions the amino group of AA tRNA in A site for attack of the peptide tRNA in P site to make peptide bond


EF-Ts restores EF-Tu (GTP)

What does Ef-Tu(GTP) do?

protein responsible for delivering all aminoacyl groups except initiation

Ef-Tu does not bind

fMet-tRNA

Ef-Tu protects

ester linkage of AA-tRNA

_____ is important for accuracy during translation elongation because _____

Ef-Tu because GTP hydrolysis is coupled to tRNA deposition, only if correct anticodon interaction occurs

Steps of translation elongation translocation

Ef-G uses GTP hydrolysis to move peptide tRNA from A site to P site


The tRNA and mRNA move


the uncharged tRNA moves to the E site and dissociates

what are the stop codons?

UGA, UAA, UAG

What does release factor 1 do?

decodes stop codons

What does release factor 3 do?

assists release factors 1 and 2

Steps of translation termination

Stop codon enters A site


RF1/2 bind


RF3 unfolds RF1/2 to bridge gap between the stop codon and PTC


Water attacks ester bond on peptidyl tRNA, releasing peptide


EF-G (GTP) and RRF dissociate 70s ribosome, tRNA and mRNA are released

GTP used in translation

fMet-tRNAf deposition by IF2-GTP


AA-tRNA deposition by Ef-TU (GTP)


Movement of ribosome by EF-G (GTP)


Release of polypep chain by RF3 (GTP)


Dissociation of ribosome by EF-G (GTP)

how do the rRNAs contribute to protein synthesis

16s: shine and delgarno sequence


23s: peptidyl tranferase center

Describe eukaryotic ribosomes

difference size (40s and 60s)

Eukaryotic translation initiator tRNA

has special tRNA that delivered by eIF-2 (GTP)


initiating Met is not formylated

Eukaryotic identification of AUG for translation initiation

depends on eIF-4E scanning from CAP to find first AUG. eIF4E is a helicase

Eukaryotic termination of translation

single release factor used

Eukaryotic mRNA structure

the translating mRNA is circular, the eIF-4E binds the cap and eIF-4E bind eIF-4G that bind the poly A tail, a circular structure is believed to faciliated reinitatiaion

Pro or Euk


One RNA may have multiple translation intiation events

pro

Pro or Euk


transcription and translation are coupled

Pro

Pro or Euk


Trancription and translation are uncoupled

Euk

Pro or Euk


Each RNA has one tranlation intiation event

euk

Where are secreted and membrane proteins translated

ER

Many antibiotics function by

inhibiting translation

2 examples of antibiotic functions that inhibit translation

Bind to 30s subunit to interfere with binding of fMet-tRNA




Analogue of an Aa-tRNA binds the A site, attaches to the growing chain, terminates translation

How are proteins targeted to the Er

The signal peptide

what is the signal peptide

9-12 hydrophoic stretch of AA, mostly at the N terminus of a secreted membrane protein

How does the signal peptide work

the signal recognition particle bidns the newly emerged signal sequence SRP is a GTP binding ribonucleoprotein with GTPase activity


Binding of SRP to the ribosome halts protein synthesis by blocking EF association and directs the complex to the ER

Where is the signal peptide sequence?

In the 1st amino acids during translation so signal to ER to finish translation in ER ribosome port, finished product in lumen

What type of proteins are translated in the ER

Seretory and membrane proteins


`

What is the signal peptide?

9-12 hydrophobic stretch of AA, mostly at the N terminus of a secreted membrane protein

Why is the signal peptide hydrophobic?

do it can interact with the inter membrane protein

With is the signal recognition particle?

Binds newly emerged signal sequence

SRP has

GTP binding ribonucleoprotein with GTPase activity


Binding of SRP to the ribosome halts protein synthesis by blocking EF association and directs the complex to the ER

The SRP receptor delivers _____ to ____ binding of the ribosome ______ that _______

The SRP ribosome SRP receptor delivers the ribosome RNA complex to the translocon, a protein conducting channel


Binding of the ribosome causes GTP hydrolysis that opens the translocon and allows protein synthesis to resume


Hydrolysis of GTP bound to the SRP allows dissociation and SRP recycling

What happens to the signal peptide once in the lumen

sometimes signal peptidases will remove it

Ferrtin

intracellular Iron storage form to keep from reaching toxic levels

Transferrin

is an iron transporter in the blood

Transferrin receptor

a membrane protein that binds iron bound transferring and facilitates its entry into the cell

Cis acting element regulating iron storage

Stem loop structure in RNA (iron response element)


present in RNA and bound by protein which effects translation of mRNA

Trans acting factor regulating iron storage

Iron response element binding protein


Binds RNA in the absence of iron


similar to lac repressor

The ferritin mRNA contains

a stem loop structure in its 5' untranslated region called the iron response element

What happens to ferreting RNA during no iron conditions

IRE binding protein binds the IRE and blocks translation

Mechanism to block translation in ferretin

IRE binding protein binds and prevents it from being scanned by helicase for AUG

Ferritin mRNA in high iron

IRE binding protein binds iron which blocks RNA binding IRE no longer bind to iRE so translation occurs

how does initiation occur in eukaryotes

recognition of CAP

The transferrin receptor mRNA has

several IREs in the 3' untranslated region

what does the transferrin receptor do?

brings iron into the cell if too much iron in cell, the cell will down regulate production of the transferrin receptor

tranferrin receptor mRNA when no iron

IRE binding protein binds and the mRNA is translated

tranferrin receptor mRNA when plus iron

IRE binding protein dissociates causing mRNA degradation

What are siRNAs

associated with mRNA degradation and loss of protein accumulation following the expression of doubled stranded RNAs

What happens when you add double stranded RNA complementary to the intron?

doesn't have effect because they're removed before entering the cytoplasm only sequences in mature mRNA are a target for this RNA degradation

What happens she injected sense stranded for locomotion gene in worms

nothing

What happens when injected antisense strand for locomotion gene in worms

nothing

what happens when injected sense and antisense strand for locomotion in worms

paralysed

Mechanism of repression by siRNA

Double stranded RNA is recognized by dicer, an RNase III enzyme


Dicer processes RNAs to 21 nucleotides


One strand of the siRNAs binds an RISC complex


The siRNA guides the risc complex to the mRNA where the siena displays complete complementarity and causes mRNA turnover

miRNAs

small RNAs encoded by the genome which are bound by risc complexes, often demonstrate parietal complementarity with mRNA resulting in translational repression

A single miRNA can

regulate many genes due to partial complementarity needed for translational repression

The microRNA binds _____ and ____

to mRNA with incorrect base pairing and forms P bodies so they cannot be translated because initiation factors are blocked

How does the IRE binding protein both increase and decrease translation of mRNA

the outcome to RNA depends on the position of its binding site


at the 5' end of the mRNA blocks scanning and at the 3' end protects from degradation

Would siRNAs that hybridize to introns catalyze mRNA degradation

no for an siena to degrade the mRNA the complementary sequence must be present in the mature mRNA

how does sanger sequencing work?

4 polymerization reactions are set up each includes the DNA to be sequences, a radio labeled primer (dan oiligo), DNA polymerase, dNTPs and a small amount of 2',3' dideoxylanalog of one of the deoxynucleoside triphosphates


Incorporation of the ddNTP analog occurs infrequently but when incorporated stops further polymerization


fragments are then separated in gel electrophoresis

PCR requires

a target DNA, primers, and heat stable polymerase

PCR consists of 3 steps per cycle

1. Heat denaturation of target DNA to generate single strands (SS)


2. annealing of primers to ss DNA at 55 degrees C'


3. DNA polymerization at 72 degrees C allowing the polymerase to synthesize DNA

Each PCR cycle _____

doubles the amount of DNA produced after n cycles 2n fold amplification

PCR cycles are repeated ____

20 to 30 times

Steps for quantitative PCR

convert mRNA into copy DNA using reverse transcriptase


add cDNA to reaction with SYBR green


SYBR green binds DNA and fluoresces which is detected


as DNA levels increase so does fluorescence


The relative mRNA level is defined by the number of cycles needed to reach an arbitrary level of fluorescence which is in the linear range of amplification (cycle threshold)



for quantitative PCR higher mRNA levels need

fewer cycles to reach the cycle threshold