Protein Synthesis, Processing, and Degradation

Protein synthesis ends at the

C-terminus

When protein is misfolded this leads to

ER stress and increase in PEK (a kinase) that phosphorylates eIF2 that prevents it from combining the a charged tRNA with the small ribosome to initiate translation.

What does the larger ribosomal subunit do?

Joins the amino acid to the polypeptide chain

What happens if the A site, P site, or E cite are blocked?

Protein synthesis will be inhibited.

The small ribosomal subunit does what?

Reads the mRNA

eIF2 does what?

Recognizes the initiating tRNAi^met and positions it on the small ribosomal subunit

Prokaryote translation is oriented using the

Shine-Delgarno sequence, positioning the mRNA on the small ribosome.

They bind to small bacterial ribosomal subunit and interfere with the binding of incoming aminoacyl~tRNA to the "A" site. The elongation phase of bacterial protein synthesis is inhibited.

Tetracycline

In eukaryotes, each of the 20-different aminoacyl tRNA synthetases recognizes

one amino acid and ALL of its cognate tRNAs

List the major components needed for the translation of protein from mRNA

1. amino acids 2. Three RNA products mRNA tRNA rRNA 3. Ribosomes 4. ATP and GTP to provide energy for the process 5. Other translation proteins

Differences between prokaryotic and eukaryotic ribosomes:

1. size 2. Since prokaryotes lack nucleus, translation in prokaryotes beings prior to the completion of transcription, while eukaryotic translation cannot occur until the transcript is transported from the nucleus to the cytosol through the nuclear pore (Gated transport)

How much energy is required to charge the tRNA (or activate the tRNA with an amino acid)?

2 high energy bonds from ATP are required (ATP is hydrolyzed to AMP). This creates an energy-rich bond.

How many sites can tRNA bind to the ribosome?

3 sites: the A site, P site, and E site

Describe in which direction a ribosome reads mRNA

5' to 3'

These amino acids are found in helices

A,E, L alanine, glutamate, leucine

What is the A site of a ribosome?

Accepts the incoming aminoacylated tRNA

tRNA structure

All have a similar secondary and tertiary structure. the acceptor end links the 5' to 3' ends to allow the formation of a structure composed of 3 hair-pin loops that have the appearance of a three-leaf clover

As the polypeptide chain emerges through the exit tunnel of the ribosome, the protein begins to spontaneously [?] and [?] into a 3-dimensional structure with [?] and [?] structures. While the gene dictates the [?] sequence of the polypeptide, additional steps and modifications may be required before it is a functional protein.

As the polypeptide chain emerges through the exit tunnel of the ribosome, the protein begins to spontaneously coil and fold into a 3-dimensional structure with secondary and tertiary structures. While the gene dictates the primary sequence of the polypeptide, additional steps and modifications may be required before it is a functional protein.

What is the large ribosomal subunit used for?

Contains the ribosomal peptidyl transferase (ribozyme) that catalyzes peptide bond formation, a translocation domain, and a tunnel through which the nascent peptide is threaded.

List the sites of protein synthesis

Cytosol

Describe the genetic code

Degenerate and unambiguous

tRNAS proofing mechanism

Ensures that the appropriate linkage to a specific tRNA is made to the appropriate amino acid. If the correct amino acid is not paired with the correct tRNA, the enzyme removes them and starts over

[?] is a bacteriostatic antibiotic that inhibits bacterial growth, particularly at higher concentrations. The drug interacts with the large subunit of the bacterial ribosome and sterically hinders the exit tunnel through which the nascent polypeptide chain moves out of the ribosome. − In resistant strains, a single base (A) of the rRNA is methylated and erythromycin no longer binds the large ribosomal subunit.

Erythromycin is a bacteriostatic antibiotic that inhibits bacterial growth, particularly at higher concentrations. − The drug interacts with the large subunit of the bacterial ribosome and sterically hinders the exit tunnel through which the nascent polypeptide chain moves out of the ribosome. − In resistant strains, a single base (A) of the rRNA is methylated and erythromycin no longer binds the large ribosomal subunit.

Where does the peptide bond formation energy come from?

From the high energy bond between the tRNA and the amino acid.

eIF 2 is a [blank] protein, regulated by a [blank] and a [blank]

G protein, regulated by a guanine nucleotide exchange factor and a GTPase

The amino acids that create turns

G, P (glycine and proline)

How does mrNA transported to the cytoplasm from the nucleus?

Gated transport via the nuclear pore

What's different int he mitochondria?

Has it's own apparatus for protein synthesis, including tRNA, RNA polymerase aminoacyl synthetases and ribosomes. Undergo a slightly different translation process.

Under low energy state what happens to protein synthesis

It elevates AMP which activates AMP kinase which phosphorylates mTOR which leads to translation being blocked because unable to proceed with the phosphorlyation of 4E-BP1 (eiF4) remains sequestered.

What happens at the P site of a ribosome?

It holds the tRNA and possesses the ribosomal Peptidyl transferase to form the peptide bond on the growing protein.

How is eIF2 regulated?

It is activated by guanine nucleotide exchange factor (Replacing the GDP with a GTP) and inactivated by a GTPase.

What is a ribozyme in the ribosome?

It is in the large subunit and it possesses the peptidyl transferase catalytic activity, used to link amino acids together.

Desribe the wobble code

It is the ability of some tRNAs to interact with more than one codon. The first two bases predominate in tRNA selection, while the 3rd base in a codon (the 3' end) can often be two or more bases (U,C,AG). Degeneracy of the genetic code means that many amino acids have more than one tRNA to which they can attach and some tRNAs can bind to more than one codon that codes for the same amino acid.

What is the E site on the ribosome?

It temporarily holds the deacylated tRNA until it Exits the ribosome

Polyribosomes (polysomes)

Many ribosomes may associate with a single mRNA to translate the mRNA at a time. This saves both time and resources. It allows the cell to make more protein more quickly and with less mRNA. It is rare that only a single protein molecule would need to be made

What is mTOR?

Mechanistic Target of rapamycin. It is a nutrient and growth factor sensor in many cells. It is a serine/threonine kinase. It is necessary for protein synthesis to proceed.

Protein synthesis starts at the

N-terminus

[blank blank] recognize the stop codon by binding to the [blank] site of the ribosome in a way that mimics [blank] and uses the [blank] of GTP to induce a [blank] change causing the [blank] of a nascent peptide.

Release factors recognize the stop codon by binding to the A site of the ribosome in a way that mimics tRNA and uses the hydrolysis of GTP to induce a conformational change causing the release of a nascent peptide.

Resources are scarce Under [?] energy conditions, such as [?] concentrations of AMP, the AMP Kinase (AMPK) is [?], and it [?] mTOR. The 4E-BP1, binding protein, remains [?] and a key component of eIF4 (eIF4E) remains [?] and translation is [?]

Resources are scarce Under low energy conditions, such as high concentrations of AMP, the AMP Kinase (AMPK) is activated, and it inhibits mTOR. The 4E-BP1, binding protein, remains unphosphorylated and a key component of eIF4 (eIF4E) remains sequestered and translation is blocked

Termination occurs when the [?] site of the [?] advances to one of [?] stop codons on the mRNA. An [?] does not bind to a stop codon. Instead the codons are recognized by [? ?] (?).? ? with the help of _[blank]__ [blank] the completed protein from the final [?] in the [?] site. Following the[?] of two [?] to [?] the nascent polypeptide chain is released from the [?] in the [?]-site. The mRNA is then[?] , and the large and small ribosomal subunits [?]. The ribosomal components along the tRNA can be [?] for additional protein synthesis.

Termination occurs when the A site of the ribosome advances to one of three stop codons on the mRNA. An amino-tRNA does not bind to a stop codon. Instead the codons are recognized by release factors (RFs). Peptidyl transferase with the help of RFs hydrolyze the completed protein from the final tRNA in the P site. Following the hydrolysis of two GTPs to GDPs the nascent polypeptide chain is released from the tRNA in the P-site. The mRNA is then released, and the large and small ribosomal subunits dissociate. The ribosomal components along the tRNA can be recycled for additional protein synthesis.

The "loaded" small ribosome (loaded with pre-initiation complex) uses the ATP-dependent [blank] on [blank] complex to scan the [blank] in a 5' to 3' direction until the initiating [blank] codon is identified. Once the start codon is identified the [blank] on the [blank] is hydrolyzed to GDP. The initiating factors then[blank] and the [blank] ribosomal subunit joins the complex to form a functional ribosome.

The "loaded" small ribosome (loaded with pre-initiation complex) uses the ATP-dependent helicase on eIF4 complex to scan the 5' untranslated region (UTR) in a 5' to 3' direction until the initiating AUG codon is identified. Once the start codon is identified the GTP on the eIF2 is hydrolyzed to GDP. The initiating factors then dissociate and the large ribosomal unit joins the complex to form a functional ribosome.

How does the eIF4 complex help the mRNA prepare for the small ribosome subunit attachment?

The ATP-dependent helicase in the eIF4 complex begins to unwind the mRNA allowing attachment of the small ribosome subunit with the correct 5' to 3' orientation

Where is the ER targeting signal sequence?

The amino-termus of the protein

The first step in elongation is forming a [blank] bond. The [blank] group on the [blank]-end terminus adds with the [blank] group of the incoming amino acid. The appropriately charged tRNA is brought to the [blank] site based on the [blank] codon. Using the enzyme ribosomal [blank blank] plus the [blank] of [blank]. The [blank] then advances [blank] nucleotides towards the [blank] end of the mRNA. This process is called [blank]. Once the new amino acid has been [blank] attached, the empty [blank] is moved to the [blank] site. This continues and repeats until it encounters a [blank] codon. It occurs at a rate of [blank] amino acids/second. Each cycle requires [blank] high energy bonds. [blank] from ATP used during [blank] and [blank] from GTP used by the [blank blank] factors (blank and blank)

The first step in elongation is forming a peptide bond. The carboxyl group on the amino-end terminus adds with the amino group of the incoming amino acid. The appropriately charged tRNA is brought to the A site based on the mRNA codon. Using the enzyme ribosomal peptidyl transferase plus the release of water. The ribosome then advances 3 nucleotides towards the 3'-end of the mRNA. This process is called translocation. Once the new amino acid has been covalently attached, the empty tRNA is moved to the E site. This continues and repeats until it encounters a stop codon. It occurs at a rate of 3-5 amino acids/second. Each cycle requires 4 high energy bonds. 2 from ATP used during a.a. activation and 2 from GTP used by the protein elongation factors (eIF1 and eIF2)

What is the small ribosomal subunit responsible for?

The formation of the initiation complex, binding the mRNA to decode the genetic information and control the fidelity of codon anti-codon pairing

Where are the ribosomal subunits assembled?

The nucleolus where newly transcribed rRNAs associate with the ribosomal proteins transported into the nucleus after their translation in the cytoplasm. The subunits are then transported to the cytoplasm where they remain separate unless they combine with the mRNA and tRNA to synthesize a polypeptide.

The two active sites on a tRNA are the [blank] end with the 3' [blank blank] to bind [blank] and the hairpin loop which includes the [blank] triplet which base pairs with [blank] codons.

The two active sites on a tRNA are the 3' OH end with the CCA sequence to bind amino acids and the hairpin loop which includes the anticodon triplet which base pairs with mRNA codons.

Approximately 10% of tRNA is chemically modified in the nucleus, why?

These changes affect tRNA structure, customizing it to interact with the ribosome to ensure the fidelity of codon recognition

Where is the energy used to activate the tRNA by aminoacyl tRNA synthetase used?

To link the amino acid to the growing polypeptide chain. (Peptide bond formation)

True or False: An individual ribosome might be membrane-bound when it translates one peptide and then free for the next protein.

True

List the nucleotide sequences for the stop codons

UAA UAG UGA

Describe how the "stress response" impacts eIF2 function and apply the concept as to why-improperly folded proteins leading to ER stress would lead to eIF2 phosphorylation

Under conditions of stress, several kinases are induced that phosphorylate eIF2. eIF2 cannot bind to the guanine nucleotide exchange factor (eIF2B) , and therefore cannot be reactivated, blocking initiation and ultimately translation. This mechanism allows the cell to inhibit protein synthesis at times when the cell/body might need to conserve energy or shuttle cellular resources into other processes. In addition, a low concentration of ternary complex prompts increased activation of amino acid synthesis genes.

The amino acid populate the middle of the beta-sheets

V, I valine, isoleucine

Describe how mTOR regulates the eIF4 complex and apply this information as to why mTOR would be down-regulated when energy is low.

When resources are scarce there is an increase in AMP and that inhibits mTOR, therefore it is unable to phosphorylate 4E-BP1 which prompts the release of a component of eIF4 leaving it free to join the eIF4 complex of proteins and allowing translation to proceed.

ER-targeting signal sequence determines what?

Whether or not the ribosome will either be "free" or "membrane-bound"

While many smaller proteins will spontaneously self-assemble, proper [?] of larger proteins often requires the help of other proteins called [?]. Proteins fold in stages. Immediately upon emerging from the ribosome,[?] structures start to form. Native-like [?] structures quickly assemble into domains ([?][?]) but lack the tightly packed interior found in the final native conformation. The incompletely folded [?][?] stage has exposed [?] patches. The [?] bind to the "sticky" [?] patches on nascent polypeptide chains as it is being created by the ribosome to prevent non-productive folding pathways or aggregation from occurring. They help the protein [?] through the[?] of intermediate conformations prior to reaching the final structure. They help with the creation of complexes composed of multiple polypeptide chains and stabilize proteins as they move through intercellular organelles. [?] include a family of proteins called[? ? ?]. [? ? ] protein synthesis is a universal response to adverse conditions, such as a rise in temperature, exposure to cold or UV light, wound healing or tissue remodeling, that cause improper folding and even denaturation of proteins.[? ?] proteins are named for their molecular weight. For example, a family of 70 kilodalton heat shock proteins (Hsp70 family) is key in the cell's machinery for protein folding

While many smaller proteins will spontaneously self-assemble, proper folding of larger proteins often requires the help of other proteins called chaperones. Proteins fold in stages. Immediately upon emerging from the ribosome, secondary structures start to form. Native-like secondary structures quickly assemble into domains (molten globules) but lack the tightly packed interior found in the final native conformation. The incompletely folded molten globule stage has exposed hydrophobic patches. The chaperones bind to the "sticky" hydrophobic patches on nascent polypeptide chains as it is being created by the ribosome to prevent non-productive folding pathways or aggregation from occurring. They help the protein fold through the stabilization of intermediate conformations prior to reaching the final structure. They help with the creation of complexes composed of multiple polypeptide chains and stabilize proteins as they move through intercellular organelles. Chaperones include a family of proteins called heat shock proteins. Heat shock protein synthesis is a universal response to adverse conditions, such as a rise in temperature, exposure to cold or UV light, wound healing or tissue remodeling, that cause improper folding and even denaturation of proteins. Heat shock proteins are named for their molecular weight. For example, a family of 70 kilodalton heat shock proteins (Hsp70 family) is key in the cell's machinery for protein folding

The initiating AUG is recognized by

a special initiator tRNA (met~tRNAi^Met) and facilitated by IF-2-GTP

What enzyme covalently couples the incoming amino acid to the tRNA?

aminoacyl- tRNA synthetase with the hydrolysis of ATP to AMP (two high energy bond breakages)

The ternary complex is formed when

eIF2 binds to GTP and met-tRNAi^Met.

[?] is a [?]-protein that recognizes the initiating tRNAi-?and positions it on the [?] ribosomal subunit (?S). [?] activity is activated by a [?] nucleotide exchange factor (replacing the [?] with a [?]) and inactivated by a [?]. Under conditions of [?], several kinases are induced that phosphorylate [?]. Phosphorylated [?] cannot bind to the [?] nucleotide exchange factor (?) and, therefore, cannot be reactivated, blocking [?] and ultimately [?]. This mechanism allows the cell to [?] protein synthesis at times when the cell/body might need to [?] energy or [?] cellular resources into other processes. In addition, a [?] concentration of [?] complex prompts [?] activation of amino acid synthesis genes.

eIF2 is a g-protein that recognizes the initiating tRNAi-met and positions it on the small ribosomal subunit (40S). eIF2 activity is activated by a guanine nucleotide exchange factor (replacing the GDP with a GTP) and inactivated by a GTPase. Under conditions of stress, several kinases are induced that phosphorylate eIF2. Phosphorylated eIF2 cannot bind to the guanine nucleotide exchange factor (eIF2B) and, therefore, cannot be reactivated, blocking initiation and ultimately translation. This mechanism allows the cell to inhibit protein synthesis at times when the cell/body might need to conserve energy or shuttle cellular resources into other processes. In addition, a low concentration of ternary complex prompts increased activation of amino acid synthesis genes.

[blank] is a part of the [blank] complex and it temporarily [blanks] translation by [blank] the binding of the [blank] ribosomal subunit to the complex until the[blank blank] is identified. It also provides a [blank] site for other eIFs.

eIF3 is a part of the eIF4 complex and it temporarily inhibits translation by blocking the binding of the large ribosomal subunit to the complex until the start codon is identified. It also provides a binding site for other eIFs.

The mRNA in eukaryote translation is oriented with respect to the small subunit of the ribosome by the

eIF4 complex

[?] is a heterotrimeric complex that, together with its binding partners, connects the 5' [?] of the mRNA and [? ? ?] ([?]) to the [?] ribosomal subunit ([?]S). Its ATP- dependent [?] (a component of the [?] complex) unwinds the mRNA structure to form a stretch of single-stranded RNA allowing docking of the [?] complex (and ultimately find the initiating [?]). A subunit of [?] is an important target of [?] [(? ? ? ?)] signaling.[?] is a highly conserved [?]/[?] [?] and the major growth factor/nutrient [?] in the cell. It senses cellular nutrients, oxygen, and energy levels. It makes sense that this type of sensor would be able to modulate translational activity. When times are [?], it will [?] translation and under low energy conditions it will [?] the process down. One of the [?] targets of [?] is 4E-BP1 a binding protein that can sequester a component of [?], [?] the formation of the initiation complex ([?] translation).

eIF4 is a heterotrimeric complex that, together with its binding partners, connects the 5' cap of the mRNA and polyadenylation binding protein (PABP) to the small ribosomal subunit (40S). Its ATP- dependent helicase (a component of the eIF4 complex) unwinds the mRNA structure to form a stretch of single-stranded RNA allowing docking of the preinitiation complex (and ultimately find the initiating AUG). A subunit of eIF4 is an important target of mTOR (mechanistic Target of Rapamycin) signaling. mTOR is a highly conserved serine/threonine (Ser/Thr) kinase and the major growth factor/nutrient sensor in the cell. It senses cellular nutrients, oxygen, and energy levels. It makes sense that this type of sensor would be able to modulate translational activity. When times are flush, it will upregulate translation and under low energy conditions it will slow the process down. One of the phosphorylation targets of mTOR is 4E-BP1 a binding protein that can sequester a component of eIF4, preventing the formation of the initiation complex (inhibiting translation).

Unambigous genetic gode means

each codon will always codes for the same, specific specific amino acid

[?] and its associated pathways are areas of much clinical and translational research. These pathways appear to be disturbed in multiple disorders. Of note, [?] expression is often [?] in human cancers, resulting in increased [?] of 4E-BP1 and the release of eIF4E; this relieves translational repression, enhancing cancerogenic protein synthesis.

mTOR and its associated pathways are areas of much clinical and translational research. These pathways appear to be disturbed in multiple disorders. Of note, mTOR expression is often upregulated in human cancers, resulting in increased phosphorylation of 4E-BP1 and the release of eIF4E; this relieves translational repression, enhancing cancerogenic protein synthesis.

List the nucleotide sequences that correspond to the initiation codon

methionine AUG

The genetic code is degenerate meaning,

multiple codons code for a single amino acid

The Shine-Delgarno sequence is

purine rich sequence 6-10 bass upstream from the initiating AUG codon.

[blank] is the nucleic acid component of the ribosome (autocatayltic and structural)

rRNA is the nucleic acid component of the ribosome (autocatayltic and structural)

the molecule that carries amino acids

tRNA

What is the mRNA required for

template that provides the code for translation

Where is the amino acid added to the tRNA?

the 3' OH terminal with the consensus sequence CCA (-OH terminal)

What proteins recognize and bind the 5' cap of the mRNA and the polyadenylation binding protein (PABP) circularizing the mRNA?

the proteins in the eIF4 complex

The preinitiation complex forms when

the ternary complex (eIF2, chaged tRNA, and GTP) bind to the small ribosomal component, with the help of eIF3 and other initiation factors.