Chapter 14: Mechanisms and Regulation of Translation

I. Introduction

A. Introduction To Translation: What Is Translation?

1. In the process of gene expression, translation is the third major step

a. Transcriptionb. Pre-mRNA processing

2. The goal of translation is to produce a protein

3. The protein is what is going to perform the function of the gene

a. Assuming proper foldingb. Assuming the proper modifications are made

B. Introduction To Translation: How The Mechanisms Of Translation Were Discovered

1. Many aspects of the mechanism of translation were learned through the study of how viruses worked

2. Viruses are composed of generally two types of molecules

a. Nucleic Acidb. Protein

3. Viruses cannot reproduce on their own

a. Cannot replicate their own genetic materialb. Cannot express their own genes

4. Viruses use the host cell machinery to do both

a. Virus preferentially has the host cell translate its mRNAsb. RNA biologists figured out how translation of cellular mRNAs works

C. Introduction To Translation: The Importance of Translation

1. Translation is the most highly conserved process in nature

a. All viruses perform translation

b. All organisms perform translationc. The RNA world

2. The process of translation is an energetically costly process

a. In a rapidly growing bacterial cell, about 80% of a cell’s energy is used for translationb. 50% of a cell’s dry weight is dedicated to translation

3. The synthesis of a single protein requires the coordinated action of over 100 proteins and RNAs

a. Some are part of the ribosomeb. Some are involved in binding the ribosome to mRNAc. Some are involved in bringing amino acids to the site of translation

II. RNAs That Are Required For Translation

A. RNAs That Are Required For Translation: Eukaryotic mRNA Structure

1. As defined by the presence of the start and stop codons, the mRNA has three basic units

a. 5’ Untranslated Region (UTR)b. Open Reading Frame (ORF)c. 3’ Untranslated Region (UTR)

2. The 5’UTR:

a. Scanned by the 40S subunit of the ribosomeb. Has significant secondary structure-regulation

3. The open reading frame:

a. Also known as the protein coding regionb. Begins with the presence of a start codon on the 5’ end and ends with a stop codon on the 3’ end

4. The 3’UTR:

a. Serves as a site of mRNA regulationb. Has significant secondary structure

5. Supplemental Figure: RNAs That Are Required For Translation: Eukaryotic mRNA Structure

B. RNAs That Are Required For Translation: Eukaryotic vs. Prokaryotic mRNA

1. mRNAs can have multiple ORFs

a. Monocistronic messages (one ORF)b. Polycistronic messages (multiple ORFS)

2. Eukaryotic mRNAs

a. Most are monocistronicb. C. elegans does produce some polycistronic mRNAs

3. Prokaryotic mRNAs

a. Commonly are polycistronicb. Ex. Lac operon

C. RNAs That Are Required For Translation: mRNA Reading Frames

1. In a eukaryotic mRNA the start codon is almost always the first AUG

2. The start codon has three functions

a. Define the start site of translation b. Specifies the first amino acid to be incorporated into the new proteinc. Defines the reading frame for all subsequent codons

3. As we learned, the sequence of the mRNA is read 3 nucleotides at a time (considered codons)

4. Each codon is immediately adjacent to, but not overlapping with the next codon

a. Any stretch of mRNA could be translated in three different frames (which would lead to greatly different proteins)b. The start codon will actually show us which of the three frames is the correct one

D. RNAs That Are Required For Translation: Introduction To tRNA

1. tRNA molecules serve as the adaptors between the codons in the mRNA and the amino acids they encode

2. “tRNA looks like nature’s attempt to make RNA do the job of a protein” –Francis Crick (Cold Spring Harbor Symposium on Quantitative Biology-1966)

3. RNA chains can fold into unique 3-Dimensional structures that can act similarly to globular proteins

4. The 3-Dimensional (tertiary) structures found in RNA arise from interactions between multiple secondary structural domains

5. There have to be many different types of tRNAs, each encoded by a separate gene

a. Creates multiple tRNA with different anti-codonsb. Each of these tRNA with different anti-codons bind different codonsc. Each of these tRNA with different anti-codons are charged with different amino acids

E. RNAs That Are Required For Translation: Basic tRNA Structure

1. The first structure published for the tRNA was the cloverleaf secondary structure by R.W. Holley et. al.

a. Studied the tRNA for alanineb. Contained three loopsc. Structure is incorrect

2. tRNA twists into an L-shaped 3-dimensional structure

a. Through X-ray crystallographyb. The “loops” from the cloverleaf structure are present as stems in the actual L-shaped structure

F. RNAs That Are Required For Translation: tRNA Maturation and Modified Bases

1. The pre-tRNA is produced and then undergoes processing to get a mature tRNA

a. pre-tRNA is transcribed about twice the length of its final form –b. pre-tRNA transcript is processed by various nucleases at both the 5’ and 3’ ends to produce a tRNA that is about 76 nt long

2. tRNA also must form complex tertiary structure to function properly-to form tertiary structure:

a. Extensive base pairingb. Incorporation of modified bases which help to increase the amount of extensive base pairing within the tRNA structure

3. tRNA can also contain more than 50 modified bases-types of modifications

a. Methylation b. Restructuring of the purine ring

4. Inosine (I) was the first modified base to be identified in tRNA

5. Pseudouridine ( ) is the most commonly seen modified base Ψin tRNA

a. The first modified nucleoside seen in any RNAb. Pseudouridine has a restructured pyrimadine ring

G. RNAs That Are Required For Translation: tRNA Loops Have A Separate Function

1. The defining of the “loops” came from the initial tertiary structure-the cloverleaf

3. The tRNA has three loops

a. U loop (T-loop)Ψb. D-loopc. Anticodon Loop

4. The U loop is involved in ribosome bindingΨ

5. The anticodon loop is involved in base pairing with the codon in mRNA

a. Consists of the 3 nitrogenous bases

b. Is bounded by a uracil on the 5’ side and a modified purine on the 3’ sidec. Each 3 base codon in the mRNA specifies a specific tRNA which is charged with a specific amino acid

6. The D-loop:

a. Is recognition by the aminoacyl tRNA synthetasesb. “charge” the tRNA with the appropriate amino acid

H. RNAs That Are Required For Translation: Coaxial Stacking of Stems in The L-Shaped tRNA Structure

1. The L-shaped structure is the true structure

a. Seen by X-ray crystallographyb. The loops we talked are really stems

2. The list of stems in the L-shaped tRNA

a. The 7 base pair acceptor stem stacks on the 5 bp T-stem to form one continuous A-type helical arm of 12 bpb. The D-stem and the anticodon stem also stack

3. The two sets of stacked stems give the tRNA its L-shaped structure

4. Co-axial stacking is a common feature of RNA, for example in rRNA co-axial stacking of as many as 70 base pairs can be found

J. RNAs That Are Required For Translation: L-Shaped Model of the tRNA Structural Features

1. For each tRNA, the appropriate amino acid is directly bound to the 3’ end of the tRNA

a. Every tRNA have the sequence 5’-P-CCA-OH-3’ at the far 3’ end of the tRNAb. Correct amino-acyl tRNA synthetase will charge the tRNA based on the anticodon sequence

2. Another commonly observed motif in the tRNA is the U turn

3. The U-turn is caused by hydrogen bonding of the N3 position of uridine with the phosphate group of a nucleotide three positions downstream

K. RNAs That Are Required For Translation: Attachment of Amino Acids To The tRNA

1. Overall, there are two classes of amino-acyl tRNA synthetases

a. Class I enzymes which attach the amino acid to the 2’OH of the final nucleotide of the tRNA and are monomericb. Class II enzymes which attach the amino acid to the 3’ OH of the final nucleotide and are dimeric or tetrameric

2. Each amino acid has its own amino-acyl tRNA synthetase

L. RNAs That Are Required For Translation: Attachment of Amino Acids To The tRNA

1. When a tRNA becomes bound to an amino acid, an acyl linkage is formed between the carboxyl group of an amino acid and either the 2’ or 3’ OH group of the final nucleotide (which is an adenine)

2. The acyl linkage is considered a high energy bond

a. Hydrolysis of this bond results in a large change in free energyb. Energy released when this bond is broken during translation helps drive formation of a peptide bond between amino acids

3. The process of creating the acyl linkage between the tRNA and the amino acid occurs in two enzymatic steps

a. Adenylylation (transfer of AMP), in which the amino acid reacts with ATP to become adenylated with the release of pyrophosphateb. tRNA charging in which the adenylylated amino acid reacts with the tRNA resulting in a transfer of the amino acid to the 3’ end of the tRNA via either the 2’ or 3’ OH group of the final nucleotide and a release of AMP

III. The Ribosomome

A. The Ribosome: An Introduction

1. The ribosome is a macromolecular machine that directs the synthesis of proteins

2. The ribosome consists two subunits in both prokaryotes and eukaryotes

a. Small Subunitb. Large Subunit

3. The ribosome has an overall molecular weight of greater than 2.5 megadaltons

a. At least 3 different rRNAsb. More than 50 different proteins

4. Compared to DNA and RNA polymerase II, the ribosome actually works quite slowly

a. Adds only 2-20 amino acids per secondb. DNA polymerase can add 200-1000 nucleotides per secondc. RNA polymerase II can transcribe 30-60 nucleotides per second

5. Subcellular localization of ribosomes

a. In eukaryotes in the cytoplasm (transcription and translation are separated)b. In prokaryotes-free floating in the cell ((co-transcriptional)

B. The Ribosome: Eukaryotic vs. Prokaryotic Ribosome Structure

1. Each ribosomal subunit has a particular function

a. The large subunit which contains the peptidyl transferase center which is responsible for formation of peptide bondsb. The small subunit which contains the decoding center in which charged tRNAs decode the information in the mRNA codon by codon

2. By convention, the large and small subunits are named according to their velocity of sedimentation when centrifuged

3. As measured by Svedbergs (S), the prokaryotic ribosome sediments the following way

a. Small subunit sediments at 30S

b. Large subunit sediments at 50Sc. Total ribosome sediments at 70S

4. As measured by Svedbergs (S), the eukaryotic ribosome sediments the following way

a. Small subunit sediments at 40Sb. Large subunit sediments at 60Sc. Total ribosome sediments at 80S

C. The Ribosome: RNA Composition of The Large and Small Subunits

1. Like the full subunits, the rRNAs are distinguished by the centrifugal speed at which they sediment in a gradient

2. The small subunits contain the following rRNAs and proteins

a. Bacterial 30S subunit contains the 16S rRNA and 21 proteinsb. Eukaryotic 40S subunit contains the 18S rRNA and 33 proteins

3. The large subunits contain the following rRNAs and proteins

a. Bacterial 50S subunit contains the 5S rRNA and 23S rRNA and 34 proteinsb. Eukaryotic 60S subunit contains the 5S rRNA, the 5.8S rRNA and the 28S rRNA as well as 49 proteins

D. The Ribosome: Functions of rRNA

1. Locations of rRNA and proteins in each each ribosomal subunit

a. rRNA located at the core-suggests important roleb. Proteins at the periphery

2. Main functions of the rRNA in the large subunit

a. Scaffold from which to build the larger superstructure of each subunitb. May have catalytic function as the peptidyl transferase center in the large subunit is composed almost entirely of rRNA

3. rRNA function in the small subunit

a. Anticodon loops of the charged tRNAs contact the 16S rRNA in bacteriab. The small subunit interacts with the mRNA through the 16S rRNA

E. The Ribosome: tRNA Binding Sites In The Small Subunit

1. The small subunit (in both prokaryotes and eukaryotes) has a total of three sites for binding tRNAs

a. A-siteb. P-sitec. E-site

2. The A-site functions to bind the amino-acylated tRNA

3. The P-site functions as the binding site for the peptidyl-tRNA (tRNA which holds the growing peptide)

4. The E-site is the binding site for the uncharged tRNA that will exit after transfer to the amino-acylated tRNA

IV. Eukaryotic Translational Initiation

A. Eukaryotic Translational Initiation: Introduction

1. The process of initiation allows for the start of translation

2. In order for translation to be initiated, three events must occur

a. The ribosome must be recruited to the mRNAb. A charged tRNA must be placed in the P-site (This is the only time a charged tRNA directly binds in the P-sitec. Positioning of the ribosome over the start codon (which allows for determination of the reading frame)

3. The tRNA that is necessary for initiation in eukaryotes is termed the Met-tRNAi

Met

B. Eukaryotic Translational Initiation: The Ribosome Is Recruited To The 5’ Cap

1. In eukaryotes, each subunit of the ribosome binds at separate times

2. The first step eukaryotic initiation is creating of the 43S pre-initiation complex

3. The two critical components of the 43S pre-initiation complex are as follows

a. 40S ribosomal subunitb. Met-tRNAi

Met bound to eIF2-GTP which is considered the ternary complex

4. The role of the eIF2-GTP is to place the Met-tRNAiMet in the P-

site of the small subunit

5. Other eukaryotic initiation factors (eIFs) that are associated with the 43S pre-initiation

a. eIF1b. eIF3 c. eIF5

C. Eukaryotic Translational Initiation: The Ribosome Is Recruited To The 5’ Cap

1. A series proteins are bound to the 5’MpppG cap that are responsible for recruitment of the 43S pre-initiation complex

a. eIF4Eb. eIF4Ac. eIF4G

2. eIF4E is considered the cap binding protein and directly binds the cap structure

3. eIF4G associates with the cap by binding eIF4E

4. eIF4A is a helicase that associates with both the mRNA as well as eIF4G, and is thought to be able to unwind secondary structure ahead of the ribosome during translation (not required)

5. eIF4G is involved in one other critical interaction when it comes to translational initiation

6. eIF4G is able to bind the poly(A) binding protein (PAB)

a. PAB is associated with the 3’ end of the mRNAb. This interaction is necessary for efficient translational initiation

7. Several observations show this interaction is necessary efficient initiation

a. Circularized mRNA have been visualized by atomic force microscopyb. Synthetic mRNAs containing a cap, but not a poly(A) tail are not efficiently translatedc. Synthetic mRNAs containing a poly(A) tail, but not a cap also are not efficiently translated

D. Eukaryotic Translational Initiation: The Small Subunit Scans The mRNA To Find The Start Codon

1. The 43S pre-initiation complex will bind the mRNA at the 5’ MpppG cap

a. Through interactions with eIF4Gb. Creates the 48S pre-initiation complex

2. Once the 43S pre-initiation complex becomes associated with the mRNA

a. Scan the mRNA in the 5’ 3’ direction for the first start codonb. Scanning is an ATP dependent process that is stimulated by the eIF4A RNA helicase

3. The start codon is recognized through base pairing between the anticodon and the initiator tRNA

a. Base pairing between the 5’ AUG 3’ start codon and the 5’ CAU 3’ anticodonb. Reason why initiator tRNA is part of the 43S pre-initiation complex

E. Eukaryotic Translational Initiation: Start Codon Context Is Important

1. Recognition of the start codon by the small ribosomal subunit is dependent on sequence context

2. The optimal sequence around the first 5’-AUG- 3’ for initiating translation was discovered by Marilyn Kozak and is known as the Kozak sequence

3. The Kozak sequence is as follows 5’-G/ANNAUGG-3’

a. The first base in the Kozak sequence needs to be a purine and is 3 bases upstream from the first nucleotide in the start codonb. The final base in the Kozak sequence is a guaninec. Not all mRNAs have this consensus sequence, but those that do are more efficiently translated

4. The Kozak sequence is thought to guide the interaction between the anticodon of the initiator tRNA and the start codon of the mRNA

F. Eukaryotic Translational Initiation: Joining Of The Large Ribosomal Subunit

1. As 43S pre-initiation complex reaches the start codon several reactions must occur to allow 60S subunit joining (contains peptidyl transferase center)

a. Correct base pairing between the initiator tRNA and the codon changes the conformation of the 43S pre-initiation complexb. This change in conformation of the 43S complex results in a change in conformation of eIF5 which then stimulates eIF2 to hydrolyze its GTP to GDP

2. eIF2 hydrolysis of GTP results in the dissociation of the following proteins

a. eIF2-GDPb. eIF1c. eIF3d. eIF5

3. In the process of dissociation of several eIFs, eIF5B-GTP binds the initiator tRNA

4. The role of eIF5B-GTP is to then stimulate the joining of the 60S ribosomal subunit

5. The joining of the 60S subunit results in the formation of the 80S ribosome

6. Once the 80S ribosome is formed, eIF5B-GTP hydrolysis is stimulated leading to release of the rest of the translational initiation factors

7. At this point the 80S initiation complex is formed

a. A full functional ribosome is now bound to the mRNA at the start codonb. The initiator tRNA is bound in the P-sitec. The start codon is positioned at the P-site

V. Translational Elongation

A. Translational Elongation: Introduction

1. Once the 80S ribosome is assembled, translation has been initiated

2. The process of polypeptide synthesis is considered translational elongation

3. During translational elongation two general events must occur

a. Peptide bonds must form between the carboxy terminal amino acid residue in the growing peptide and the new amino acid to be addedb. The ribosome must translocate

4. The ribosome incorporates 2-20 amino acids per second, and so it moves about 2-20 codons per second along the mRNA

B. Translational Elongation: The Peptidyl Transferase Reaction

1. The reaction to form a new peptide bond is the peptidyl transferase reaction and is catalyzed by the large subunit of the ribosome

2. In the peptidyl transferase reaction requires two components

a. Amino-acylated tRNAb. Peptidyl tRNA-3’ end is attached to the carboxyl terminus of the growing peptide

3. To catalyze new peptide bond formation, the 3’ ends of the amino-acyl and peptidyl tRNAs are brought in close proximity

a. Allows the amino group of the amino acid attached to the amino-acyl tRNA to attack the carbonyl group of the carboxy terminal amino acid attached to the peptidyl tRNAb. Allows a new peptide bond to form

4. There are two important consequences of this method of peptide synthesis

a. Allows for synthesis of the peptide in the amino to carboxyl terminal directionb. Allows transfer of the growing peptide from the peptidyl tRNA to the amino-acyl tRNA

C. Translational Elongation: Introduction To Incorporation Of The Correct Amino Acid

1. Translational elongation adds amino acids to the growing peptide

2. Three events that occur to allow incorporation of the correct amino acid

a. The correct amino-acyl tRNA must be placed within the A-site of the ribosome (as directed by the codon which is lying in the A-siteb. A peptide bond must be formed between the amino acid linked to the tRNA in the A-site (amino-acyl tRNA) and the growing peptide linked to the tRNA in the P-site (peptidyl tRNA) through a peptidyl transferase reactionc. The tRNA in the A-site must be translocated to the P-site to allow for the next amino-acyl tRNA to bind

3. In addition, translocation will allow for a new codon to enter the A-site

4. Unlike initiation, which requires many different proteins, translational elongation only requires two proteins

a. eEF1 (composed of two subunits) (works like prokaryotic EF-TU)b. eEF2 (composed of multiple subunits) (works like prokaryotic EF-G)

D. Translational Elongation: Delivering The tRNA To The A-site

1. eEF1 role in elongation:

a. Binds an amino-acylated tRNAb. Brings amino-acylated tRNA to ribosome

2. eEF1-protein function is dependent on the state of the guanine nucleotide bound to it

a. Guanine nucleotide state ensure that only a charged tRNA enters the A-siteb. eEF1-GTP is capable of being bound to amino-acylated tRNAc. eEF1-GDP is not capable of being bound to amino-acylated tRNA

3. Any eEF1-GTP bound amino-acyl tRNA can enter the A-site of the ribosome whether its anticodon base pairs with codon in the mRNA or not

a. Attempted base pairing between anti-codon and codonb. If an amino-acylated tRNA enters the A-site with the wrong anticodon, it just dissociates from the A-site

4. A series of events happens once the amino-acylated tRNA with the correct anti-codon base pairs with the codon,

a. First, base pairing between the anticodon and codon occurs, eEF1 hydrolyzes its GTP to GDPb. eEF1-GDP dissociates from the amino-acyl tRNA in the A-sitec. eEF1-GTP hydrolysis is a way of “proofreading” or ensuring the correct amino acid is added to the growing peptided. Not the final proofreading mechanism

E. Translational Elongation: Another Proofreading Mechanism Exists After eEF1 Is Released

1. To participate in the peptidyl transferase reaction, the tRNA must rotate into the peptidyl transferase of the large subunit (accomodation)

a. During the accomodation process, the 3’ end of the amino-acylated tRNA moves about 70 Angstromsb. Incorrectly base paired tRNAs cannot rotatate and dissociate from the A-site (second proofreading mechanism)

2. If the correct tRNA is in the A-site, it is able to appropriately rotate

a. Allows the amino group of the amino acid to be added to come close to the carboxy terminus of the growing peptide bound to the tRNA in the P-siteb. The amino group attacks the carboxy group of the last

amino acid in the growing peptide chain allowing a new peptide bond to formc. At this point, the growing peptide is transferred to the next tRNA

F. Translational Elongation: Translocation of The Ribosome

1. When a new peptide bond forms, the ribosome need to translocate one codon 3’ on the mRNA

a. Moves the next codon into the A-site and causes the A-site of the ribosome to become emptyb. Moves the previous codon into the P-site (along with the new peptidyl tRNA)c. Moves the empty tRNA into the E-site

2. Translocation of both subunits does not happen at the same time

a. The large ribosomal subunit which catalyzes peptide bond formation translocates before the small ribosomal subunitb. This leads to the 3’ ends of each tRNA being shifted into their new locations, but their anticodon ends are still in their pre-peptide bond locations

3. The initial steps of translocation are coupled with the peptidyl transferase reaction

4. eEF2 catalyzes translocation

a. Will only bind the ribosome in the GTP bound state

b. eEF2 has roughly has the same structure as a protein bound tRNA

5. Steps in ribosome translocation

a. Large subunit shiftsb. The shift of the large ribosomal subunit will leave part of the A-site in the large ribosomal subunit empty and available for eEF2-GTP bindingc. Upon binding, GTP hydrolysis is stimulatedd. GTP hydrolysis causes a change in conformation of eEF2, which allow it to enter the part of the A-site which is present in the small ribosomal subunit, displacing the tRNA present theree. Upon entry of eEF2-GDP into the part of the A-site in the small ribosomal subunit, translocation of the A-site tRNA is triggered allowing the ribosome to fully move 1 codon 3’ along the mRNA

6. When translocation is complete, the ribosomal structure has markedly reduced affinity for eEF2-GDP, which leads to its release

7. Supplemental Figure: Translational Elongation: Translocation of The Ribosome (Molecular Mimicry)

G. Translational Elongation: Forming A Peptide Bond Is An Energy Dependent Process

1. Translational elongation is a very energy dependent process

2. For every peptide bond, the following is consumed

a. 1 molecule of ATP (used to charge the tRNA and create the acyl linkage)b. 2 molecules of GTP (during the elongation process)

3. Note: The energy from GTP is spent on ensuring accuracy during the elongation process, and propelling the ribosome down the mRNA in the 5’ 3’ direction

VI. Translational Termination

A. Termination of Translation: Introduction

1. Termination of translation occurs when a stop codon enters the A-site of the ribosome

a. UAAb. UAGc. UGA

2. The two events necessary for termination to occur

a. Release of the peptideb. Dissociation of the ribosome from the mRNA

3. The proteins that are involved in peptide release are considered release factors (RFs) and there are two classes of these

a. Class 1 release factors decode the stop codonsb. Class 2 release factors are GTPases that stimulate the activity of the class 1 release factors

B. Termination of Translation: The Mechanism

1. Termination begins when the stop codon enters the A-site

2. Note: There is no tRNA that recognizes a stop codon

3. Instead, the stop codon is recognized by eRF1

a. Is a class 1 factor that is bound to GTPb. Can recognize all stop codons

4. Once eRF1 recognizes the stop codon, eRF3 mediates hydrolysis of the eRF1-GTP to eRF1-GDP

a. Hydrolysis of the acyl linkage between the carboxy terminal amino acid and the peptidyl tRNAb. Release of eRF1 from the ribosomal A-sitec. Mechanism of action is unknown

VII. Regulation of Translation

A. Regulation of Translation: Introduction

1. Gene expression can still be controlled even if an mRNA is produced, as it can be controlled at the level of translation

a. If an mRNA is translated then the gene is said to be expressedb. If an mRNA is not translated, then the gene is said to be not expressed

2. In general, a significant amount of regulation occurs at the transcriptional level

3. However, regulation of gene expression at the translational level has several advantages

a. Allows rapid responses to external stimulib. Allows for the rapid start of gene expression after periods of dormancy

4. Translational regulation mostly occurs at the level of initiation, such that synthesis of incomplete proteins does not occur

B. Regulation of Translation: Introduction To The Regulators

1. Two types of molecules have the capability of functioning to regulate translation

a. Proteinsb. RNA

2. The two types of RNA that function to regulate translation are as follows:

a. miRNA (which directly repress translation of an mRNA)b. siRNA (which repress translation of an mRNA by causing its degradation)

3. More often than not, a translational regulator will bind the mRNA in one of two places

a. 5’ Untranslated Region (5’ UTR)b. 3’ Untranslated Region (3’ UTR)

4. Binding of a regulator to the 5’ UTR results in a direct inhibition of translational initiation

a. Blocking 43S Pre-initiation complex bindingb. Blocking 40S Ribosome scanning

5. Binding of a regulator to the 3’ UTR results in inhibition of translation in ways yet to be fully understood

C. Regulation of Translation: Regulation of Ferretin mRNA

1. Regulating iron levels in the human body is of critical importance,as many proteins need to coordinate an iron ion to function properly

a. Hemoglobinb. Myoglobinc. Oxidative phosphorylation enzymes

2. Low cellular levels of iron lead to a condition called anemia, which is more common in women than in men

a. Fatigueb. Weaknessc. Shortness of breathd. Light headednesse. Palpitations

3. Severe symptoms include

a. Chest pain (up to heart attack)b. Dizzinessc. Faintingd. Rapid Heart Rate

4. Common risk factors are:

a. Poor dietb. Intestinal disordersc. Menstruation d. Pregnancye. Chronic conditions

5. Treatment involves diet changes and iron supplements

6. In response to iron levels, the translation of the ferritin mRNA is regulated

7. The ferritin mRNA (and gene) encodes the ferritin protein

a. Ferritin protein is an iron binding protein

b. Ferritin protein is a major regulator of cellular iron levelsc. Ferritin stores and releases iron in a controlled manner to maintain iron homeostasis in the cell

8. Translation of ferritin mRNA is regulated by a protein known as the iron regulatory protein (IRP)

9. IRP binds an element within the 5’ UTR of the ferritin mRNA called the Iron Response Element (IRE)

a. The IRE forms a hairpin loopb. This hairpin loop must be resolved by eIF4A in order for 43S pre-initiation complex scanning to occur

10. The ability of the IRP to recognize the IRE is controlled by the levels of iron in the cell

11. Under conditions of low concentrations of iron:

a. Iron concentration is too low and iron cannot bind the IRPb. IRP is able to bind the IRE and inhibit the ability of eIF4A to unwind the IRE hairpin structure, which in turn blocks the progression of the 43S pre-initiation complex from scanning the ferritin mRNA for the start codon

12. Under conditions of high concentrations of iron

a. Iron concentration is high, and can bind the IRPb. IRP, when bound to iron cannot bind the IREc. eIF4A can resolve the IRE secondary structure allowing 43S pre-initiation complex scanningd. Ferritin mRNA translatione. Ferritin protein acts to reduce iron levels by storing it

D. Regulation of Translation: Introduction To Global Regulators of Translation

1. Besides being able to regulate the translation of a single mRNA, translation of mRNA can be regulated in a global manner (almost all mRNA present in the cell)

2. In general, translation is globally regulated in response to two conditions

a. Reduced nutrientsb. Physiological stress

3. In these instances two early steps in translational initiation are targeted for inhibition

a. Recognition of the mRNA by the 43S pre-initiation complexb. Initiator tRNA binding to the 40S ribosomal subunit

E. Regulation of Translation: Global Regulation of Translation-eIF2 Phosphorylation

1. One common mechanism to globally inhibit translation is to phosphorylate eIF2

a. eIF2-GTP bind the initiator tRNA and delivers it to the P-site of the 40S ribosomal subunitb. eIF2-GDP cannot bind the initiator tRNA

2. eIF2 is a multisubunit protein (heterotrimer)

a. subunitαb. subunitΒc. subunitγ

3. Phosphorylation of the subunit is mediate by several kinase αenzymes

4. eIF2 kinases are activated in response to amino acid αstarvation, viral infection and elevated temperature

5. Phosphorylation of the subunit inhibits the action of a GTP-αexchange factor for eIF2 called eIF2B

a. Exchanges the GDP for GTPb. Allows eIF2 protein to engage in another round of translational initiation

F. Regulation of Translation: Global Regulation of Translation-eIF4E

1. To globally inhibit translational initiation the cell can target the 5’ cap binding protein eIF4E

a. eIF4E is required to build a cap binding complex including eIF4A and eIF4G

b. eIF4G is necessary for mRNA circularization and 43S pre-initiation complex binding

2. The domain that eIF4G uses to bind eIF4E is also found in a small family of proteins known as eIF4E binding proteins (4E-BPs)

3. The 4E-BPs act to inhibit translation globally by competing with eIF4G for binding to eIF4E

4. The activity of 4E-BPs, like many other proteins, is regulated by a phosphorylation cycle

a. Unphosphorylated 4E-BPs have the ability to bind eIF4E tightly, thus blocking in the ability of eIF4G to bind and stimulate translationb. Phosphorylated 4E-BPs do not have the ability to bind to eIF4E

5. Phosphorylation of 4E-BPs is mediated by a key cellular protein kinase called mTOR

6. The mTOR is activated in response to a receipt of a specific signal

a. Growth factorsb. Hormonesc. Factors that stimulate cell division

7. In response to these signals mTOR phosphorylates 4E-BPs leading to increased translational capacity in the cell

8. Given the fact that mTOR phosphorylation leads to increased translational capacity and increased cell division, mTOR activity can lead to cancer formation

9. Rapamycin is an mTOR inhibitor and an affective chemotherapy agent

G. Regulation of Translation: 4E-BPs Can Regulate Translation of Specific mRNAs

1. One 4E-BP named CUP regulates translation of the Oskar mRNA during Drosphila development

2. The Oskar mRNA has elements in its 3’ UTR called Bruno Response Elements (BREs) which are bound by the Bruno protein

3. Bruno in turn acts to recruit CUP protein

4. The CUP protein interacts with eIF4E to block the binding of eIF4G