Lecture 25

Translation (protein synthesis)

5’3’

From messenger RNA to protein:

The information encoded in the DNA is transferred to messenger RNA and then decoded by the ribosome toproduce proteins.

• Process of making proteinsproteins from info on mRNAmRNA– mRNA travels out of

nucleus to the ribosome, which “reads” the mRNA as a series of 3 letter words called codons

Translation

• Translation is the production of a protein using the information that is coded in the mRNA molecule

• In the cytoplasm a ribosome attaches to one end of the mRNA molecule. using a sequence that is the binding site for the ribosome

Second mRNA base

Fir

st

mR

NA

ba

se

(5 e

nd

)

Th

ird

mR

NA

ba

se

(3 e

nd

)

Genetic Code

The Code

• 64 codons

•Genetic code–redundant but not ambiguous; no codon specifies more than one amino acid (but one amino acid may have >1 codon)

•Codons– must be read in the correct reading frame in order for the specified polypeptide to be produced

Evolution of the Genetic Code

• Genetic code– nearly universal: shared by the simplest bacteria, plants,

fungi and animals

• Genes can be transcribed and translated after being transferred from one species to another

Mechanism of Translation

• Ribosomes- Bind messenger (mRNA)- Attract transfer RNA (tRNA) to mRNA – tRNA covalently linked to specific amino acid (aa-tRNA)

-Complementary basepairs form between mRNA and aa-tRNA (codon-anticodon interactions)

-Enzyme in ribosome catalyzes peptide bond between amino acids- -> polypeptide chain grows

Transfer RNA

• Transfer RNA molecules are short RNAs that fold into a characteristic cloverleaf pattern. Some of the nucleotides are modified to become things like pseudouridine and ribothymidine.

• Each tRNA has 3 bases that make up the anticodon. These bases pair with the 3 bases of the codon on mRNA during translation.

• Each tRNA has its corresponding amino acid attached to the 3’ end. A set of enzymes, the “aminoacyl tRNA synthetases”, are used to “charge” the tRNA with the proper amino acid.

• Some tRNAs can pair with more than one codon. The third base of the anticodon is called the “wobble position”, and it can form base pairs with several different nucleotides.

Generalized tRNA tRNA structure

~ 80 nt long

Two-dimensional structure

Three-dimensional structure

Chemistry of tRNA Charging with Amino Acid

tRNAs are activated by amino-acyl tRNA synthetases

Amino acid Aminoacyl-tRNAsynthetase (enzyme)

Pyrophosphate

Phosphates

tRNA

AMP

Aminoacyl tRNA(an “activatedamino acid”)

Amino-acyl tRNA synthetases:

One synthetase for each amino acida single synthetase may recognize multiple tRNAsfor the same amino acid

Two classes of synthetase. Different 3-dimensional structuresDiffer in which side of the tRNA they recognize and how they bind ATP

Class I - monomeric, acylates the 2’OH on the terminal riboseArg, Cys , Gln, Glu, Ile, Leu, Met, Trp Tyr, Val

Class II - dimeric, acylate the 3’OH on the terminal riboseAla, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, Thr

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

One mechanism for maintaining high fidelity of proteinsynthesis is the high fidelity of aa-tRNA synthetases

Two levels of control to ensure that the proper amino acidis incorporated into protein: 1) Charging of the proper tRNA

2) Matching the tRNA to the messenger RNA

Incorporation of amino acids into polypeptide chains

Incorporation of the correct amino acyl-tRNA is determinedby base-pairing interactions between the anticodon of the tRNA and the messenger RNA

Protein synthesis occurs on ribosomes

Protein synthesis occurs on ribosomes

Ribosomes

tRNAmolecules

Exit tunnelGrowingpolypeptide

Largesubunit

mRNA 3

Computer model of functioning ribosome

Smallsubunit

5

E P A

• Facilitate specific coupling of anticodons with codons• Ribosomal structure

– Two ribosomal subunits (large and small) • Made of proteins (ribosomal proteins) and ribosomal RNA (rRNA)

Form binding sites for mRNA and aa-tRNA

Structure of a ribosome

and mitochondria

S=Sedimentation coefficient

Ribosome Assembly

The proteins of each ribosomal subunitare organized aroundrRNA molecules

16S rRNA

23S rRNA secondary structure

Ribosome Assembly: takes place largely in a specialized domain ofthe nucleus, the nucleolus

In the nucleolus, RNA polymerase I transcribes the rDNA repeatsto produce a 45S RNA precursor

The 45S precursor is processedand cleaved intomature rRNAs andribosomal proteinsthen bind to generatethe large and smallribosomal subunits

3D organization of the eukaryotic large subunit rRNA

Ribosomal Proteins decorate the surface of the ribosome

Large subunit. Grey = rRNA Gold = ribosomal proteins

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

Ribosomal proteins often have extensions that chain into the core of the rRNA structure

Crystal structure of L19 L15 (yellow) positioned in a fragmentof the rRNA (red)

Mitochondrialor Prokaryotic

Eukaryotic 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 bindingsites:A site = amino-acyltRNA binding site

P site = peptidyl-tRNAbinding site

E site = exit site

In addition to the APE sites there is an mRNA binding groovethat holds onto the message being translated

There is a tunnel through the large subunit that allows thegrowing polypeptide chain to pass out of the ribosome

Mechanism of protein synthesis

Protein synthesis falls into three stages .

1.initiation-the assembly of a ribosome on an mRNA molecule.

2.elongation-repeated cycles of amino acid addition.

3.termination-the release of the new protein chain.

•All three stages require protein translation factors

prokaryotic eukaryotic functionInitiation factor

IF1 IF3

IF2

eIF3 eIF4c eIF6 eIF4B eIF4F

eIF2B eIF2

eIF5

Bind to ribosome submits

Bind to mRNA

Initiator tRNA delivery

Displacement of other factors

Elongation factor

EF-Tu

EF-Ts

EF-g

eEF1α

eEF1βγ

eEF2

Aminoacyl tRNA delivery

Recycling of EF-Tu or eEF1α

Translocation

Termination factors

RF1, RF2, RF3 eRF Polypeptides Chain release

Initiation is controlled differently in prokaryotic and eukaryotic ribosomes

In prokaryotes a single transcript can give rise to multiple proteins

Initiation

Polyribosomes (exists in prokaryotes and viruses)

• a single mRNA (transcript) is translated by many ribosomes simultaneously

• mRNA+ bound ribosomes= polyribosomes (polysome)

• Allows fast synthesis of many copies a polypeptide

RNA polymerase

DNA

Polyribosome

RNApolymerase

Direction oftranscription

mRNA

0.25 m

DNA

Polyribosome

Polypeptide(amino end)

Ribosome

mRNA (5 end)

Messenger RNAs are translated on polyribosomes

Initiation (prokaryote)

In prokaryotes, initiation requires • the large and small ribosome

subunits,• the mRNA • the initiator tRNA • three initiation factors .

• In prokaryotes, ribosomes bind to specific translation initiation sites. There can be several different initiation sites on a messenger RNA: a prokaryotic mRNA can code for several different proteins. Translation begins at an AUG codon, or sometimes a GUG. The modified amino acid N-formyl methionine is always the first amino acid of the new polypeptide.

• In eukaryotes, ribosomes bind to the 5’ cap, then move down the mRNA until they reach the first AUG, the codon for methionine. Translation starts from this point. Eukaryotic mRNAs code for only a single gene. (Although there are a few exceptions, mainly among the eukaryotic viruses).

• Note that translation does not start at the first base of the mRNA. There is an untranslated region at the beginning of the mRNA, the 5’ untranslated region (5’ UTR).

Initiation

Initiator tRNA

• Methionine is the first amino acids incorporated into a protein chain in both prokaryotes (modified to N-formylmethionine) and eukaryotes.

• Initiator tRNAs are special tRNAs recognizing the AUG (Alternative start codon GUG) start codons in prokaryotes and eukaryotes.

• Initiator tRNAs differ from the one that inserts internal Met residues.

Initiator tRNA formation in E. coli

1. Both initiator tRNA and noninitiator tRNAmet are charged with Met by the same methionyl-tRNA synthetase to give the methionyl-tRNA

2. Only the initiator methionyl-tRNA is modified by transformylase to give N-formylmethionyl-tRNAfmet.

Size comparisons show that the ribosome is large enough to bind tRNAs and mRNA.

Met

GTPInitiator tRNA

mRNA

53

mRNA binding site

Smallribosomalsubunit

Start codon

P site

5 3

Translation initiation complex

E A

Largeribosomalsubunit

GDP

Met

• The initiation process involves first joining the mRNA, the initiator methionine-tRNA, and the small ribosomal subunit. Several “initiation factors”--additional proteins--are also involved. The large ribosomal subunit then joins the complex.

Initiation (prokaryote)

Schematic model showing binding sites on ribosome

P site (Peptidyl-tRNAbinding site)

E site (Exit site)

mRNAbinding site

A site (Aminoacyl-tRNA binding site)

Largesubunit

Smallsubunit

E P A

The assembled ribosome has one exit site and two tRNA-binding sites, which are called A- and P-site, for aminoacyl and peptidyl sites respectively.

Only fMet-tRNAfMet can be used for initiation by 30S subunits; all other aminoacyl-tRNAs are used for elongation by 70S ribosomes.

In prokaryotes, specific sequences in the mRNA aroundthe AUG codon, calledShine-Delgarno (SD) sequences,are recognized by an intiationcomplex consisting of a Metamino-acyl tRNA, Initiation Factors (IFs) and the smallribosomal subunit

GTP hydrolysis by IF2 coincident with release of the IFs and binding of the largeribosomal subunit leads to formation of a completeribosome,on the mRNAand ready to translate.

IF1 and IF3 bind to a free 30S subunits.

IF2 complexed with GTP then bind to the small subunits, forming a complex at RBS.

The initiator tRNA can then bind to the complex at the P site paired with AUG codon.

The 50S subunits can now bind. GTP is then hydrolyzed and IFs are released to give the 70S initiation complex

30S initiation complex

Most of the differences in the mechanism of protein between prokaryotes and eukaryotes occur in the initiation stage, where a greater numbers of eIFs and a scanning process are involed in eukaryotes.

The eukaryotic initiator tRNA does not become N-formylated.

Initiation (eukaryote)

The initiation factor can be grouped to there function as follow

Binding to ribosomal subunits eIF6 eIF3 eIF4c

Binding to the mRNA eIF4B eIF4F eIF4A eIF4E

Involved in initiation tRNA delivery

eIF2 eIF2B

Displace other factors eIF5

Initiation (eukaryote)

In contrast to the events in prokaryotes, initiation involves the initiation tRNA binding to the 40S subuits before it can bind to the mRNA. Eukaryotic mRNA binding to the ribosomes is facilitated by the 7-methyl guanosine cap

Eukaryotic mRNAs have a distinct structure at the 5’ end

Structure of the 7-methyl guanosine cap

The 7me-G cap is requiredfor an mRNA to be translated

In contrast, Eukaryotesuse a scanning mechanismto intiate translation.

Recognition of the AUGtriggers GTP hydrolysisby eIF-2

GTP hydrolysis byeIF2 is a signal forbinding of the largesubunit and beginningof translation

Initiation (eukaryote) Scanning

The eukaryotic 40s ribosome submit complex bind to the 5’cap region of the mRNA and moves along it scanning for an AUG start codon.

Eukaryotic ribosomes migrate from the 5’ end of mRNA to the ribosome binding site, which includes an AUG initiation codon.

Elongation

With the formation of the 70S initiation complex, the elongation cycle can begin.

Elongation involves the three factors, EF-Tu, EF-Ts, EF-G, as well as GTP, charged tRNA and the 70S initiation complex.

The protein synthesis elongation cycle in prokaryotes and eukaryotes is quite similar.

The factors EF-Tu EF-Ts EF-G have direct eukaryotic equivalents called eEF1α eEF1βγ eEF2

Ribosome Association and Initiation of Translation

1. Small ribosomal subunit binds mRNA and special initiator tRNA (met-tRNAi)

(carries the amino acid methionine)

2. Small subunit scans along the mRNA until first start codon (AUG).

3. Initiation factors bring in large subunit

initiator tRNA occupies the P site.

Polypeptide

tRNA withamino acidattached

Ribosome

tRNA

Anticodon

35

mRNA

Aminoacids

Codons

Elongation

- Amino acids are added one by one to the preceding amino acid

-Elongation factors facilitate

- codon recognition

- peptide bond formation

- translocation

Amino end

mRNA

5

3

Growing polypeptide

Next amino acidto be added topolypeptide chain

tRNA

Codons

Schematic model with mRNA and tRNA

E

Ribosome translates 5’ to 3’ on mRNA.Polypeptide chain grows amino end first, carboxyl end last.

The ribosome then moves down one codon. The now-empty tRNA at the P site is displaced off the ribosome

The process is then repeated: the tRNA at the P site holds the peptide chain, and a new tRNA binds to the A site.the peptide chain is transferred onto the amino acid attached to the A site tRNA.the ribosome moves down one codon, displacing the empty P site tRNA and moving the tRNA with the peptide chain from the A site to the P site.

Elongation

Ribosome ready fornext aminoacyl tRNA

mRNA

5

Amino endof polypeptide

E

Psite

Asite

3

2

2 GDP

E

P A

GTP

GTP

GDP

E

P A

E

P A

1. Recognition

2. Peptide bondformation

3. Translocation

The three steps of elongation

1.Charged tRNA is delivered as a complex with EF-Tu and GTP .

2.Peptidyl tranferase (50S ribosomal subunit) makes a peptide bond by joining the two adjacent amino acid without the input of more energy.

3.Translocase (EF-G), with the energy from GTP, moves the ribosome one codon along the mRNA, ejecting the uncharged tRNA and transferred the ribosome peptide from the mRNA.

EF-Tu-Ts exchange cycle

A new tRNA, corresponding to the next codon on the mRNA, binds to the A site. The ribosome catalyzes a transfer of the amino acid from the P site onto the amino acid at the A site, forming a new peptide bond.

Peptide bond formation takes place by reaction between the polypeptide of peptidyl-tRNA in the P site and the amino acid of aminoacyl-tRNA in the A site.

Peptide bond formation is catalyzed by the large subunit rRNA

Translocation

• In bacteria, the discharged tRNA leaves the ribosome via another site, the E site. • In eukaryotes, the discharged tRNA is expelled directly into the cytosol.• EF-G (translocase) and GTP binds to the ribosome, and the discharged tRNA is ejected from the P-site in an energy consuming step.• the peptidyl-tRNA is moved from A-site to P-site and mRNA moves by one codon relative to the ribosome

Proper reading of theanticodon is the secondimportant quality controlstep ensuring accurateprotein synthesis

=EF-1 Elongation factors Introduce a two-step“Kinetic proofreading”

A second elongation factorEF-G or EF-2, drives the translocation of the ribosome along the mRNA

Together GTP hydrolysisby EF-1 and EF-2 help driveprotein synthesis forward

• Occurs when stop codon in mRNA reaches A site of ribosome

• Three codons are called “stop codons”. They code for no amino acid, and all protein-coding regions end in a stop codon.

• A site accepts protein called release factor • When the ribosome reaches a stop codon, there is

no tRNA that binds to it. Instead, proteins called “release factors” bind, and cause the ribosome, the mRNA, and the new polypeptide to separate. The new polypeptide is completed.

• Release factor causes addition of water molecule instead of amino acid

Termination

• Note that the mRNA continues on past the stop codon. The remaining portion is not translated: it is the 3’ untranslated region (3’ UTR).

3

The release factor hydrolyzes thebond between the tRNA in theP site and the last amino acid of thepolypeptide chain. The polypeptideis thus freed from the ribosome.

The two ribosomal subunitsand the other componentsof the assembly dissociate.

Releasefactor

Stop codon(UAG, UAA, or UGA)

5

3

5

3

5

Freepolypeptide

When a ribosome reaches a stopcodon on mRNA, the A site of theribosome accepts a protein calleda release factor instead of tRNA.

Termination

Protein factors called release factors interact with stop codon and cause release of completed polypeptide chain.

RF1 and RF2 recognizes the stop codon with the help of RF3

The release factors make peptidyl transferase transfer the polypeptide to water, and thus the protein is released

Release factors and EF-G: remove the uncharged tRNA and release the mRNA.

Termination (prokaryote)

Termination (eukaryote)

Eukaryotes use only one release factors eRF, which requires GTP,recognize all three termination codons.

Termination codon is one of three (UAG, UAA, UGA) that causes protein synthesis to terminate.

Release Factor is a molecular mimic of a tRNA

eRF1 tRNA

TRANSCRIPTION

RNA PROCESSING

RNAtranscript

5

Exon

NUCLEUS

FORMATION OFINITIATION COMPLEX

CYTOPLASM

3

DNA

RNApolymerase

RNA transcript(pre-mRNA)

Intron

Aminoacyl-tRNAsynthetase

Aminoacid

tRNA

AMINO ACID ACTIVATION

3

mRNA

A

P

E Ribosomalsubunits

5

Growingpolypeptide

E A

Activatedamino acid

Anticodon

TRANSLATION

Codon

Ribosome

Post-Translational Modification• New polypeptides usually fold themselves spontaneously into

their active conformation. However, some proteins are helped and guided in the folding process by chaperone proteins

• Many proteins have sugars, phosphate groups, fatty acids, and other molecules covalently attached to certain amino acids. Most of this is done in the endoplasmic reticulum.

• Many proteins are targeted to specific organelles within the cell. Targeting is accomplished through “signal sequences” on the polypeptide. In the case of proteins that go into the endoplasmic reticulum (ER), the signal seqeunce is a group of amino acids at the N terminal of the polypeptide, which are removed from the final protein after translation.

Targeting Polypeptides to Specific Locations

In eukaryotes, what are the two populations of ribosomes?

Free, soluble in cytosol synthesize soluble proteins

Bound to rER- synthesize secreted or membrane bound proteins

- tagged with signal peptide at amino end

Ribosomes

mRNASignalpeptide

Signal-recognitionparticle(SRP)

SRPreceptorprotein

CYTOSOL

ER LUMEN Translocationcomplex

Signalpeptideremoved

ERmembrane

Protein

Signal peptide targets polypeptides to ERfinal polypeptide destined for secretion or membrane

Is the molecular weight of a secreted protein different than the predicted translation product of its mRNA?

Effect of mutations on gene expression

Any change in the genetic material

What is a mutation?

Types of mutationsPoint: a single nucleotide change

-substitution gcca->gcga

-deletion gcca->gca

-insertion gcca->gacca

• From spontaneous mutations: occur during DNA replication, recombination, or repair

• From mutagens are physical or chemical agents that can cause mutations

Source of Mutations

Wild-type hemoglobin DNA

3 5 53

Mutant hemoglobin DNA

Normal hemoglobin Sickle-cell hemoglobin

What kind of mutation? substitution

Substitutions

• Missense mutations– Change codon to encode a different amino acid

• Nonsense mutations– Change codon to encode a stop codon

nearly always leading to a nonfunctional protein

Missense mutations are more common.

Base-pair substitution

No effect on amino acid sequenceU instead of C

MissenseA instead of G

NonsenseU instead of A

Stop

Amino end

Protein

5 3

Carboxyl end

Stop

Stop

Stop

mRNA

Wild type

Neutral

Change in amino acid

Premature termination

Substitutions

Insertions and Deletions

• Alters reading frame ->frameshift mutation

• Often more devastating than substitutions

Ribosomal Frameshifting

Because translationuses a triplet code,there are three potentialreading frames in each mRNA

As the ribosome translocates, it moves in three nucleotidesteps, ensuring that the frame defined by the AUG is usedthroughout translation

If the ribosome moves 1 or 2 (or 4 or 5) nucleotidesthis produces a frameshift

Many retroviruses induce ribosomal frameshifting in the synthesis of viral proteins

e.g. HIV

Protein synthesis is often regulated at the level of translation initiation

General control of translational initiation

ie. all transcripts of the cell are effected (though the relativeeffect differs between specific mRNAs)

Global downregulation or upregulation can occur in response to various stimuli the most common are

1) Nutrient availabilitylow 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 by eIF2 phosphorylation

Stimulated byAmino acid deprivation

Control of translation by eIF4E availability

The 7MEG cap binding subunit of eIF4, eIF4E, is sequesteredby eIF4E binding proteins (4E-BPs). The binding of theseproteins is regulated by their phosphorylation state

GrowthFactors

NutrientLimitation

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

Ferritin is a cytosolic iron binding protein expressed wheniron 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, bycontrol of protein synthesis.

Regulation by iron (Fe):

Inhibitors of Protein Synthesis

• Those that affect prokaryotic but not eukaryotic protein synthesis are effective antibiotics

• Streptomycin - an aminoglycoside antibiotic - induces mRNA misreading. Resulting mutant proteins slow the rate of bacterial growth

• Puromycin - binds at the A site of both prokaryotic and eukaryotic ribosomes, accepting the peptide chain from the P site, and terminating protein synthesis

Translation Inhibitors are important antibiotics

Sample questions

• The site of protein synthesis is

• A. Ribosome

• B. Nucleus

• C. Endoplasmic reticulum

• D. Chromosome

Sample questions

• The structure in a bacterium that indicates an active site for protein synthesis is

• A. a chromosome.

• B. a cell membrane,

• C. a flagellum.

• D. a polysome.

Sample questions

• Which of the following is not necessary for protein synthesis to occur, once transcription is completed?

• A. tRNA• B. Ribosomes• C. mRNA• D. DNA

Sample questions

• During the process of translation:

• A. the peptide is ‘passed’ from the tRNA in the P-site to the tRNA in the A-site.

• B. incoming tRNAs must first bind to the E-site.

• C. initiation begins with the binding of the ribosomal SSU to the poly-A tail of the mRNA.

• D. the mRNA is translated by one ribosome at a time.

• The nucleolus of the nucleus is the site where:

• A. RNA processing occurs

• B. rRNA is transcribed and ribosomal subunits are assembled

• C. tRNA are charged with amino acids

• D. mRNA is translated into protein

• The ribosomes are composed of

• A. proteins

• B. RNA

• C. both (a) and (b)

• D. lipids

• Which is required for protein synthesis?

• A. tRNA

• B. mRNA

• C. rRNA

• D. All of these

• In the genetic code there are:

• A. more tRNAs than codons.• B. more codons than amino acids.• C. more nucleotides than codons. • D. the same number of codons and amino acids

• he anticodon of tRNA

• A. binds to rRNA• B. binds to an amino acid• C. binds to the Shine Dalgarno sequence• D. binds to an mRNA codon

• Initiation of eukaryotic translation begins when the:

• A. large and small subunits link together, then bind to the mRNA.

• B. ribosomal small subunit holding an initiator tRNA binds to the 5’ end of mRNA.

• C. ribosome binds to of the start codon and an initiator tRNA enters the ribosome.

• D. initiator tRNA binds to the start codon, followed by binding of the ribosome large subunit.

• On the ribosome, mRNA binds

• A. between the subunits• B. to the large subunit• C. to the small subunit• D. none of these

• The peptidyl transferase reaction occurs

• A. on the large subunit

• B. on the small subunit

• C. between the subunit

• D. none of these

• Ribosomes select the correct tRNAs

• A. based on the aminoacyl group

• B. solely on the basis of their anticodons

• C. depending on their abundance in the cytosol

• D. with the least abundant anticodons

• Which of the following amino acid starts all proteins synthesis?

• A. Glycine

• B. Proline

• C. Thymine

• D. Methionine

• The growing polypeptide chain is released from the ribosomes when

• A. a chain terminating codon is reached

• B. a chain terminating tRNA binds to the ribosome

• C. the 7-methyl guanosine cap is reached

• D. the poly A tail is reached

What is a gene?

• A gene is a region of DNA whose final product is either a polypeptide or an RNA molecule