Lects. 16&17 Translation

8/9/2019 Lects. 16&17 Translation

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Translation: From messenger RNA to protein:

The information encoded in the DNA is transferred to

messenger RNA and then decoded by the ribosome to

produce proteins.

5¶-ATGCCTAGGTACCTATGA-3¶

3¶-TACGGATCCATGGATACT-5¶

5¶-AUGCCUAGGUACCUAUGA-3¶

5¶-AUG CCU AGG UAC CUA UGA-3¶

N-MET-PRO-ARG-TYR-LEU-C

Transcription

decoded as

Translation

Protein

Alanine tRNA

Generalized tRNA

Modified Bases

Found in tRNAs

tRNAs are activated by amino-acyl tRNA synthetases

Structure of an amino acyl-tRNA synthetase bound to a tRNA

One mechanism for maintaining high fidelity of protein

synthesis is the high fidelity of aa-tRNA synthetases

Amino-acyl tRNA synthetases:

One synthetase for each amino acid

a single synthetase may recognize multiple tRNAs

for the same amino acid

Two classes of synthetase.Different 3-dimensional structures

Differ in which side of the tRNA they recognize

and how they bind ATP

Class I - monomeric, acylates the 2¶OH on the terminal ribose

Arg, Cys , Gln, Glu, Ile, Leu, Met, Trp Tyr, Val

Class II - dimeric, acylate the 3¶OH on the terminal ribose

Ala, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, Thr

Two levels of control to ensure that the proper amino acid

is incorporated into protein: 1) Charging of the proper tRNA

2) Matching thecognate tRNA to the

messenger RNA

Incorporation of amino acids into polypeptide chains I

Incorporation of amino acids into polypeptide chains II

Protein synthesis occurs on ribosomes

and mitochondria

Ribosome Assembly

The proteins of each

ribosomal subunitare organized around

rRNA molecules

16S rRNA

Ribosome Assembly: takes place largely in a specialized domain of

the nucleus, the nucleolus

In the nucleolus, RNA polymerase I transcribes the rDNA repeats

to produce a 45S RNA precursor

The 45S precursor

is processed

and cleaved intomature rRNAs and

ribosomal proteins

then bind to generate

the large and small

ribosomal subunits

23S rRNA secondary structure

3D organization of the eukaryotic large subunit rRNA

Ribosomal Proteins decorate the surface of the ribosome

Large subunit. Grey = rRNA Gold = ribosomal proteins

Ribosomal proteins often have extensions that snake into

the core of the rRNA structure

Crystal structure of L19 L15 (yellow) positioned in a fragment

of the rRNA (red)

The ribosomal proteins are important for maintaining the

stability and integrity of the ribosome, but NOT for catalysis

ie. the ribosomal RNA acts as a ribozyme

Mitochondrial

or ProkaryoticEukaryotic 60S subunit 80S ribosome 40S subunit

The large and small subunits come together to form the ribosome

The association of the large and small subunits creates the

structural features on the ribosome that are essential for

protein synthesis

Three tRNA binding

sites:

A site=

amino-acyltRNA binding site

P site = peptidyl-tRNA

binding site

E site = exit site

In addition to the APE sites there is an mRNA binding groove

that holds onto the message being translated

There is a tunnel through the large subunit that allows the

growing polypeptide chain to pass out of the ribosome

Peptide bond formation is catalyzed by the large subunit rRNA

Incorporation of the correct amino acyl-tRNA is determined

by base-pairing interactions between the anticodon of the

tRNA and the messenger RNA

Proper reading of the

anticodon is the second

important quality control

step ensuring accurate

protein synthesis

Elongation factors

Introduce a two-step

³Kinetic proofreading´

A second elongation factor

or EF-2, drives thetranslocation of the ribosome

along the mRNA

Together GTP hydrolysis

by EF-1 and EF-2 help drive

protein synthesis forward

Termination of translation

is triggered by stop codons

Release factor enters

the A site and triggers

hydrolysis the peptidyl-tRNA

bond leading to release of the protein.

Release of the protein causes

the disassociation of the

ribosome into its constituent

subunits.

R l F i l l i i f RNA

Release Factor is a molecular mimic of a tRNA

eRF1 tRNA

Initiation of Translation

Initiation is controlled differently in prokaryotic and

eukaryotic ribosomes

In prokaryotes a single transcript can give rise to multiple proteins

In prokaryotes, specific

sequences in the mRNA aroundthe AUG codon, called

Shine-Delgarno sequences,

are recognized by an intiation

complex consisting of a Met

amino-acyl tRNA, InitiationFactors (IFs) and the small

ribosomal subunit

GTP hydrolysis by

IF2 coincident withrelease of the IFs and

binding of the large

ribosomal subunit leads

to formation of a complete

ribosome,on the mRNAand ready to translate.

Eukaryotic mRNAs have a distinct structure at the 5¶ end

Structure of the 7-methyl guanosine cap

The 7me-G cap is required

for an mRNA to be

translated

In contrast, Eukaryotes

use a scanning mechanism

to intiate translation.

Recognition of the AUG

triggers GTP hydrolysis by eIF-2

GTP hydrolysis by

eIF2 is a signal for

binding of the large

subunit and beginning

of translation

Messenger RNAs are translated on polyribosomes

Protein synthesis is often regulated at the

level of translation initiation

An example of control of specific mRNAs: regulation by iron (Fe):

Ferritin is a cytosolic iron binding protein expressed when

iron is abundant in the cell.

Transferrin receptor is a plasma membrane receptor important

for the import of iron into the cytosol.

They are coordinately regulated, in opposite directions, by

control of protein synthesis.

Regulation by iron (Fe):

There is also general control of translational initiation.

ie. all transcripts of the cell are effected (though the relative

effect differs between specific mRNAs)

Global downregulation or upregulation can occur in response

to various stimuli the most common are1) Nutrient availability

low nutrient (amino acids/carbohydrate)

downregulates translation

2) Growth factor signals.

stimulation of cell division upregulates translation

General control of translational initiation is exerted through

two primary mechanisms.

Control of the phosphorylation of eIF2

Control of the phosphorylation of eIF4 binding proteins

Control of translation b eIF2 phosphor lation

Control of translation by eIF2 phosphorylation

Stimulated by

Amino acid deprivation

Control of translation by eIF4E availability

The7ME

G cap binding subunit of eIF4, eIF4E, is sequestered by eIF4E binding protiens (4E-BPs). The binding of these

proteins is regulated by their phosphorylation state

GrowthFactors

NutrientLimitation

Nutritional signals can control both the recognition of the mRNA

and loading of the 40S subunit.

Nutritional

controls2

Modification of the translation machinery is a common

feature of viral life cycles

e.g. Picornaviruses

Polio virusEncephalomyocarditis virus

Picornaviruses have single stranded RNA genomes.

Poliovirus Life Cycle

The poliovirus genome is translated into a single,

large polyprotein that then auto-proteolyzes itself into

smaller proteins.

One of these proteins, viral protease 2A cleaves

the translation initiation factor eIF4G so that it

can no longer function as a bridge between themethyl cap binding subunit and the 40S subunit

The consequence of this cleavage is that translation of

cellular mRNAs stops

But«the viral RNA is still translated due to the presence of

an internal ribosomal entry site (IRES). This acts like a

bacterial initiation site to allow Cap-independent initiation

from internal AUG codons.

What is X?

³X´ is not a protein, as suggested

by the textbook model at right,rather it is a structure in the mRNA

itself that can bind to the remaining

fragment of eIF4G

Some cellular mRNAs are also translated using IRESs

During G2/M phase of the cell cycle, translation is generally

downregulated by activation of 4E-BPs. Many proteins expressed

during this period bypass this control by using IRES elements

Ribosomal Frameshifting

Because translation

uses a triplet code,there are three potential

reading frames in

each mRNA

As the ribosome translocates, it moves in three nucleotide

steps, ensuring that the frame defined by the AUG

is usedthroughout translation

If the ribosome moves 1 or 2 (or 4 or 5) nucleotides

this produces a frameshift

Many retroviruses induce ribosomal frameshifting in

the synthesis of viral proteins

e.g. HIV

Translation Inhibitors are important antibiotics

Lects. 16&17 Translation

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