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Protein Synthesis,

Processing andRegulation

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Translation

-the synthesis of proteins as directed by mRNA templates.

-carried out on ribosomes

-Protein synthesis is thus the final stage of geneexpression.

involves interactions between three types of RNA

molecules (mRNA templates, tRNAs, and rRNAs), as wellas various proteins that are required for translation

basic mechanics are also the same in all cells.

polypeptide chains are synthesized from the amino to

the carboxy terminusEach amino acid is specified by three bases (a codon) in

the mRNA, according to a nearly universal genetic code.

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1) mRNA transmits information present in DNA

2) tRNA - acts a bilingual translator molecule

3) Ribosomes and rRNA the workbenches for

protein synthesis4) tRNA synthetases - serve to mediate the

attachment of specific amino acids to specific

tRNA molecules.

5)amino acids

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Transfer RNAs serve as adaptors that align amino acids on the

mRNA template. Aminoacyl tRNA synthetases attach amino acids

to the appropriate tRNAs, which then bind to mRNA codons bycomplementary base pairing.

Transfer RNAs:

Anticodon- The nucleotide sequence oftransfer RNA that forms complementary

base pairs with a codon sequence onmessenger RNA.

aminoacyl tRNA synthetase- An enzymethat joins a specific amino acid to a tRNA

molecule carrying the correct anticodon

sequence.

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Figure 7.2. Attachment of

amino acids to tRNAs

Activated form

of amino acid

Anticodon

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Common structural components:

1. The 3 terminal-CCAsequence to which the aminoacid is bound. The encoded aminoacid covalently attaches to theribose of the terminal adenosine.

The other end of the moleculepossesses a 3 nucleotide sequence

called the anticodon which is theregion of the molecule that attaches

to the mRNA by complimentary basepairing.

2. A conserved cloverleaf

secondary structure

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Two-step activation of amino acids

The 3'-end

adenine of

a tRNA

This ester

bond gets

readily

hydrolyzed

unlessprotected

by a factor.

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The Ribosome:

particles composed of RNA and proteins that are the

sites of protein synthesis.

The 4-letter language in mRNA is translated

into 20-letter language in protein by

ribosome.

Ribosomes in

the cytoplasm

Ribosomes onER membrane

Three binding sites ofaa-tRNA across largeand small subunits

A passage for mRNA

A tunnel for emerging

peptide

Figure 7.4.Ribosome structure

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facilitate the coupling of the tRNA anticodons with mRNA codons.

Each ribosome has a large and a small subunit formed in the

nucleolus.

Ribosome is composed of proteins and ribosomal RNA (rRNA), the most

abundant RNA in the cell. rRNA is transcribed in the nucleus, then bind to

special proteins to form the ribosomal subunits in the nucleolus.

The large and small subunits join to form a functional ribosome only

when they attach to an mRNA molecule.

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Eukaryotes Prokaryotes

encodes a single polypeptide

(monocistric)

the 5 end is capped (with 7

methylguanusine

the 5- nontranslated region

separates the cap from the

translational initiation signal

encodes more than one

polypeptide (polycistronic)

The 5 end is not capped

initiation codons are

preceded by the a specific

nucleotide sequence called

the Shine-Delgarnosequence.

Both prokaryotic and eukaryotic mRNAs have 5 and 3 non coding sequences.

These are called the 5 UTR and 3 UTR.

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* Two amino acids are designated by single codons: methionine by AUG andtryptophan by UGG

The rest are designated by two, three, four or six codons. Multiple codons for a

single amino acid represent degeneracy in the code.

The genetic code is nearly universal. The same code words are used in all living

organisms, prokaryotic and eukaryotic.

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Codon Usual Code MitochondrialCode

UGA

AUA

AGA

AGG

Termination

Isoleucine

Arginine

Arginine

Tryptophan

Methionine

Termination

Termination

Non universal Codon Usage in Mammalian Mitochondria

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Translation of the codons of mRNA involves their direct interaction with

complementary anticodon sequences in tRNA. Each tRNA species carries a unique amino acid, and each has a specific three-base

anticodon sequence

codon-anticodon base pairing is Antiparallel

codons are read in a sequential, nonoverlapping reading frame

Anticodon and amino acid acceptor sites are located at opposite extremes of all tRNA

molecules

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Variances from standard base pairing are common in the codon-anticodon interactions.

Many amino acids can be carried by more than one tRNA species, and degenerate codons can

be read by more than one tRNA (but always one carrying the correct amino acid).

Wobble - the ability of one tRNA to recognize two or three different mRNA codons .

Occurs when the third base (5end) of the tRNA anticodon has some play or wobble, so that it can

hydrogen bond with more than one kind of a base in the third position (3 end) of the codon.

E.g.: the base U in the wobble position of a tRNA anticodon can pair with either A or G in the third

position of an mRNA codon

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Some tRNAs contain a modified base called inosine (I), which is in the

anticodons wobble position and can pair with U, C, or A in the third position of

an mRNA codon.

Thus, a single tRNA with the anticodonCCI

will recognize three mRNA

codons: GGU, GGC, or GGA all coding for glycine.

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3 Codon Base 5 Anticodon

Bases Possible

Some codons are read more effeciently by one anticodon than another. Not

all codons are used equally, some being used very rarely. Examination of many mRNAsequnces has allowed construction of codon usage tables that show that different

organisms preferentially use different codons to generate similar polypeptide

sequences.

3 Codon Base 5 Anticodon

Bases Possible

A

C

G

U

U or I

G or I

C or U

A or G or I

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The Organization of mRNAs and the Initiation of Translation

UTR - untranslated regions

polycistronic -messenger RNAsthat encode multiple

polypeptide chains

monocistronic -messenger RNAsthat encode a single polypeptide

chain.

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-both prokaryotic and eukaryotic cells, translation always initiates with

the amino acid methionine, usually encoded by AUG.

-In most bacteria, protein synthesis is initiated with a modified methionine

residue (N-formylmethionine), whereas unmodified methionines initiateprotein synthesis in eukaryotes (except in mitochondria and chloroplasts,

whose ribosomes resemble those of bacteria).

Figure 7.7. Signals for translationinitiation

Shine-Delgarno sequence -Thesequence prior to the initiation

site that correctly aligns bacterial

mRNAs on ribosomes.

Signals that identify initiation codons are different in prokaryotic andeukaryotic cells

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Figure 7.8. Overview of translation

The Process of Translation

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Translation cycle

Only two sites are occupied

at any time.

Ribosome is a ribozyme. The crucialfunctions are mostly performed by rRNAs.

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Translation factors

Role Prokaryotes Eukaryotes

Initiation IF-1, IF-2, IF-3 eIF-1, eIF-1A, eIF-2,

eIF-2B, eIF-3, eIF-4A,

eIF-4B, eIF-4E, eIF-4G,

eIF-5

Elongation EF-Tu, EF-Ts, EF-G eEF-1, eEF-1, eEF-

2

Termination RF-1, RF-2, RF-3 eRF-1, eRF-3

Table 7.1. Translation Factors

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Initiation with initiation factors

This ester bond is labile

and protected by eIF2.

In eukaryotes, small subunit

binds to the cap of mRNA and

moves to the first AUG codon.

Eukaryotic mRNA are

monocistronic due to this

initiation mechanism.

Some viral mRNA have an

internal ribosome entrysite (IRES) in the middle.

In bacteria, small subunit

binds to a Shine-Dalgarno

sequence anywhere in mRNA

to initiate translation. Thus,

bacterial mRNA are

polycistronic as they are

produced from an operon.

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It is the association of all of the

eIF4 factors that serves to bring the

mRNA to the 40S subunit..the

eIF4G interacts with the eIF3 on the40s subunit.

Then the 40S subunit with these

factors and tRNA attached scans

the mRNA for the initator (AUG)

codon.

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When the 40S ribosome complex encounters the AUG codon, the eIF5 initiates

hydrolysis of the GTP bound to eIF2.

causes eIF2 which is bound to GTP to

lose a phosphate. This means thateIF2 and any other associated factors

are now only bound to GDP.

This hydrolysis event, signals for

the release of any factor

associated with GDPtheremoval of eIF2, eIF4A, 4, eIF1A,

eIF1, eIF3, eIF4E and eIF4G

This serves as the signal to allow the 60s

Subunit to bind to the 40s Subunit. This isreferred to as the 80s initiation complex.

Once this has formed, then elongation of the protein can begin.

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Bacterial ribosomes engaged in elongating a polypeptide chain exist as 70S

particles.

Initiation of protein synthesis is not a function of intact ribosomes, but is

undertaken by

the separate subunits, which reassociate during the initiation reaction.

Initiation occurs at a special sequence on mRNA called the ribosome-binding site. Thisis a short sequence of bases that precedes the codingregion .The small and large subunits associate at the ribosome binding site

to form an intact ribosome.

The reaction occurs in two steps:

Recognition of mRNA occurs when a small subunit binds to form an

initiation complex at the ribosome-binding site.

A large subunit then joins the complex to generate a complete ribosome.

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Although the 30S subunit is involved in initiation, it is not by itself

competent to undertake the reactions of binding mRNA and tRNA.

It requires additional proteins called initiation factors (IF). These factorsare found only on 30S subunits, and they are released when the 30S subunitsassociate with 50S subunits to generate 70S ribosomes. This behavior

distinguishes initiation factors from the structural proteins of the ribosome.

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Several proteins called elongation factors take part in this three step

cycle which adds amino acids one by one to the initial amino acid:

1. Codon recognition.2. Peptide bond formation.

3. Translocation.

The ribosome as 3 sites for tRNA binding.the P or peptidyl site,

the A or aminoacyl site and E or exit site. The A, E and P sites are used overand over during elongation.

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The elongation cycle of translation - overview

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(Prokaryotes)

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1. Codon recognition

The mRNA codon in the A site of the ribosome forms hydrogen bonds

with the anticodon of an entering tRNA carrying the next amino acid in the

chain.

An elongation factor EF-Tu directs tRNA into the A site in bacteria. In

eukaryotes eEF-1 (4 subunits: eEF-1, eEF-1, eEF-1, eEF-1)

eEF-1 consists of eEF-11 and eEF-12

Hydrolysis of GTP provides energy for this step.

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2. Peptide bond formation.

A peptide bond is formed between the

polypeptide in the P site and the new amino acidin the A site by a peptidyl transferase. Thisreaction requires hydrolysis of GTP bound to EF-Tu, or eEF1.

This inactivates EF-Tu, it is ejected from the

ribosome and regenerated by EF-Ts. Noeukaryotic homology of EF-Ts is known, butpossibly one of the subunits of the eEF-1 has suchactivity.

Peptidyl transferase activity appears to be one

of the rRNAs in the large ribosomal subunit .

The polypeptide separates from its tRNA andis transferred to the new amino acid carried bythe tRNA in the A site.

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3. Translocation.

The tRNA in the A site, which is

now attached to the growing peptide, istranslocated to the P site.Simultaneously, the tRNA that was inthe P site is translocated to the E siteand from there it exits the ribosome.

During this process, the codon and

anticodon remain bonded, so thatmRNA and the tRNA move as a unit,bringing the next codon to betranslated into the A site.

The mRNA is moved through the

ribosome only in the 5 to 3 direction.

Translocation requires GTPhydrolysis and is mediated by EF-G inbacteria and by eEF-2 in eukaryotes.

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3. Translocation.

The tRNA in the A site, which is now attached to the growing

peptide, is translocated to the P site. Simultaneously, the tRNAthat was in the P site is translocated to the E site and from there itexits the ribosome.

During this process, the codon and anticodon remain

bonded, so that mRNA and the tRNA move as a unit, bringingthe next codon to be translated into the A site.

The mRNA is moved through the ribosome only in the 5 to 3direction.

Translocation requires GTP hydrolysis and is mediated by EF-G in bacteria and by eEF-2 in eukaryotes.

This process continues until a termination codon is encountered by the

ribosome.

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Proofreading in elongation

Translation does not need to be as extremely accurate as DNA replication.

Yet, there are several error correction steps in translation

This ester bond is labileand protected by EF-Tu.

All four base-pairs

have identical Hbond

patterns in

the minor grooves.

In all base-pairs,

glycosidic bond

positions are the

same, which is usedin checking correct

base-pairings.

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Termination of Translation

Typical stop codons are UAA, UAG, and UGA. When one of these

sequences is translocated into the A site, this is the signal to stoptranslation.

-Cells do not contain tRNAs with anticodons that are complimentary

to these sequences, so it is impossible for translation to continue.

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There are release factors that recognize this sequence and terminate

protein synthesis.

In eukaryotic cells, there is one release factor (eRF1) that recognizes

all 3 stop codons.

(Prokaryotes)

Termination with release factors

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The release factors

resemble a tRNA to enter

the A site and provides

an H2O molecule tohydrolyze the last tRNA

off the polypeptide

Nascent peptide moves

through a water-filled tunnel.

The walls made of large subunitrRNA are like Teflon coating

for easy sliding. Small

hydrophobic spots are embedded

in extensive hydrophilic surface

of the wall.

Bacterial mRNA aretranslated without

processing before

transcription termination.

Eukaryotic mRNA form a

circle to allow for rapid

re-binding of ribosome.

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What if you need lots of protein to carry out a

particular function within a cell?

-A single mRNA can be translated

simultaneously by several ribosomes in both

prokaryotic and eukaryotic cells.

-Once a ribosome has moved away from the

initaiton site another ribosome can bind and

start synthesis of a new polypeptide chain. This

is what you see here.

-It is common for a single mRNA to be translated

by several ribosomes spaces about 100-200

nucleotides apart. When this occurs and there

are multiple ribosoem attached to the mRNA it

is referred to as a polyribosome.

Each ribosome synthesizes its own separatepolypeptide chain.

-This allows for a much more rapid syntheis of a

given protein. Why might this be important? To

allow a cell to respond to its environment

quickly

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INHIBITOR SPECIFIC EFFECT

Acting only on bacteria

Tetracycline blocks binding of aminoacyl-tRNA to A-site of ribosome

Streptomycin prevents the transition from initiation complex to chain-elongating

ribosome and also causes miscoding

Chloramphenicol blocks the peptidyl transferase reaction on ribosomes (step 2)

Erythromycin blocks the translocation reaction on ribosomes (step 3)

Rifamycin blocks initiation of RNA chains by binding to RNA polymerase

(prevents RNA synthesis)

Acting on bacteria and eucaryotes

Puromycin causes the premature release of nascent polypeptide chains by its

addition to growing chain end

Actinomycin D binds to DNA and blocks the movement of RNA polymerase (prevents

RNA synthesis)

Acting on eucaryotes but not bacteriaCycloheximide blocks the translocation reaction on ribosomes (step 3)

Anisomycin blocks the peptidyl transferase reaction on ribosomes (step 2 )

-Amanitin blocks mRNA synthesis by binding preferentially to RNA polymerase II

The ribosomes of eucaryotic mitochondria (and chloroplasts) often resemble those of bacteria in their sensitivity to inhibitors Therefore

Table 6-3. Inhibitors of Protein or RNA Synthesis