Biochemistry Chapter 15 - Translation: Synthesis of Proteins

Missense mutation

A change that specifies a different amino acid CGA → CCA Arg → Pro

A silent mutation

A change that specifies the same amino acid CGA → CGG Arg → Arg

Synthesis of proteins on the RER

(1) Translation of the protein begins in the cytosol. (2) As the signal peptide emerges from the ribosome, a SRP (Signal recognition particle) binds to it and to the ribosome and inhibits further synthesis of the protein. (3) The SRP binds to the SRP receptor in the RER membrane, docking the ribosome on the RER. (4) The SRP is released and protein synthesis resumes. (5) As the signal peptide moves through a pore into the RER, a signal peptidase removes the signal peptide. (6) Synthesis of the nascent protein continues, and the completed protein is released into the lumen of the RER.

Aminoacyl-tRNA synthetase

1. "charging" the tRNA 2. there are 20 synthetases in most organisms, one for each amino acid 3. errors in tRNA charging will result in the wrong amino acid incorporated into polypeptides

Reduced Beta Globin: Disease is less severe and due to__________________(5)

1. Promoter mutations 2. Cap site mutations 3. Splice junction mutations 4. Missense mutations 5. RNA Cleavage/Polyadenylation mutations

How do Aminoacyl-tRNA Synthetases recognize their cognate tRNA?

1. Some recognize the Anticodon plus other Nucleotides in the tRNA 2. Some recognize only other Nucleotides in the tRNA

tRNA charging has 2 levels of specificity:

1. The tRNA anticodon and other sequences in the tRNA interact with one domain of the synthetase 2. The appropriate amino acid interacts with another domain of the synthetase near the 3' end of the tRNA

Each amino acid is carried to the ribosome by an __1___ (i.e. tRNA with an amino acid __2__ attached)

1. aminoacyl-tRNA 2. covalently

Note that the codon-anticodon pairing is __1__ and __2__.

1. complementary 2. antiparallel

Hypoxanthine (I) is an unusual base found in __1__ that can form base pairs with U, C, or A. It is formed by the deamination of adenine. Hypoxanthine is the base attached to ribose in the nucleoside _2_ .

1. tRNA 2. inosine

Proteins are produced by the process of ___1____, which occurs on ____2____ and is directed by __3__ .

1. translation 2. ribosomes 3. mRNA

translation process Follows the _________________ antiparallel rules of nucleic acid base pairing

5' -> 3' / 3' -> 5'

Nonsense mutation

A change that produces a stop codon CGA → UGA Arg → Stop

A Point mutation

A single base change

There are 3 translation termination codons

UAA, UGA, UAG

b-Thalassemia: not enough _____________

beta-Globin

A site - P site - E site -

A site -incoming aminoacyl-tRNA binding P site - peptidyl-tRNA binding E site - exit/ejection

Translation initiation occurs at an ___ codon on most mRNAs

AUG

Insertion/Deletion mutation

An addition of one or more bases/A loss of one or more bases

Post translational modification of proteins (table) ____________________ occurs in ER and Golgi

Glycosylation

The Code is Degenerate but Unambiguous, Explain what that means.

Because many amino acids are specified by more than one codon, the genetic code is described as "degenerate," which means that an amino acid may have more than one codon. However, each codon specifies only one amino acid, and the genetic code is thus unambiguous.

____________: more than one codon MAY encode a single amino acid

Degenerate

Where does methionyl-tRNAiMet bind on the ribosome?

During initiation, Met-tRNAiMet binds to the ribosome at the P site, which is located initially at the start codon for translation.

Elongation of polypeptide chain

Elongation of a polypeptide chain. (1) Binding of valyl-tRNAVal to the A site. (2) Formation of a peptide bond. (3) Translocation. (4) Ejection of the free tRNA. After step 4, step 1 is repeated using the aminoacyl-tRNA for the new codon in the A site. Steps 2, 3, and 4 fol-low. These four steps keep repeating until termination occurs. EF, elongation factor.

Synthesis of the Protein and Termination (Elongation) (three steps)

Elongation of the polypeptide involves three steps: (1) binding of an aminoacyl-tRNA to the "A" site on the ribosome where it base-pairs with the second codon on the mRNA; (2) formation of a peptide bond between the first and second amino acids; and (3) translocation, movement of the mRNA relative to the ribosome, so that the third mRNA codon moves into the "A" site. These three elongation steps are repeated until a termination codon aligns with the site on the ribosome where the next aminoacyl-tRNA would normally bind. Release factors bind instead, causing the completed protein to be released from the ribosome.

How does ribosome establish at which AUG segment will translation start? And what happens if the sequence is lost?

Eukaryotes also contain a Kozak con-sensus sequence, which is recognized by the ribosome as the translational start site (the sequence is A or G - CCAUGG, where the purine base is three bases upstream of the AUG start codon). The Kozak sequence aids in defining the initial AUG codon for translation. Loss of this sequence reduces the efficiency of translational initiation.

For each amino acid in the polypeptide, how many high-energy bonds (ATP) are cleaved?

Four high-energy bonds. Formation of each aminoacyl-tRNA requires the equivalent of two high-energy phosphate bonds because ATP is converted to AMP and pyrophosphate, which is cleaved to form two inorganic phosphates. As each amino acid is added to the growing peptide chain, two GTPs are hydrolyzed, one at the step involving eEF1A and the second at the translocation step.

If the wrong aa-tRNA is delivered to the A site, ________________ does not occur

GTP hydrolysis

When are proteins posttranslationally glycosylated?

Glycosylation, the addition of carbohydrate groups, is a common modification that occurs mainly on proteins that are destined to be secreted or incorporated into lysosomes or cellular membranes.

N-terminal signal sequences are __________________ amino acid side chains

Hydrophobic

Formation of a peptide bond. Does a separate protein conduct the formation of the bond?

In the first round of elongation, the amino acid on the tRNA in the A site forms a peptide bond with the methionine on the tRNA in the P site. In subsequent rounds of elongation, the amino acid on the tRNA in the A site forms a peptide bond with the peptide on the tRNA in the P site. Peptidyltransferase, which is not a protein but the rRNA of the large ribosomal subunit, catalyzes the formation of the peptide bond. The tRNA in the A site now contains the growing polypeptide chain, and the tRNA in the P site is uncharged (i.e., it no longer contains an amino acid or peptide).

Synthesis of the Protein (Initiation)

Initiation involves formation of a complex containing the initial methionyl-tRNA bound to the AUG "start" codon of the mRNA and to the "P" site of the ribosome. It requires guanosine triphosphate (GTP) and proteins known as eukaryotic initiation factors (eIFs).

Initiation of protein synthesis

Initiation of protein synthesis. P site, peptidyl site on the ribosome; A site, aminoacyl site on the ribosome; E site, free tRNA ejec-tion site (the A, P, and E sites or portions of them are indicated by dashed lines); eIF, eukaryotic initiation factor. This is a simplified version of translational initiation because many more initiation factors and steps are required.

tRNA: _________ has degenerate base pairing: It pairs with either Adenosine or uracil

Inosine

I-cell disease (mucolipidosis II)

Is a disorder of protein targeting. Lysosomal proteins are not sorted properly from the Golgi to the lysosomes, and lysosomal enzymes end up being secreted from the cell. This is because of a mutation in the enzyme N-acetylglucosamine phosphotransfer-ase, which is a required first step for attaching the lysosomal targeting signal, mannose 6-phos-phate, to lysosomal proteins. Thus, lysosomal proteins cannot be targeted to the lysosomes, and these organelles become clogged with materials that cannot be digested, destroying overall lysosomal function. This leads to a lyso-somal storage disease of severe consequence, with death before the age of 8 years.

ER or Golgi complex residence: ________________ sequence for return from golgi to ER

KDEL [lys-asp-glu-leu]

Eukaryotic polypeptide chain Elongation

Met-tRNAi binds to P site at initiation 1. The next aa-tRNA, escorted by eEF1A-GTP, associates with the codon in A site 2. 28S rRNA Peptidyltransferase catalyzes peptide bond formation. The growing polypeptide is transferred to the aa-tRNA in the A site 3. eEF2-GTP stimulates ribosome translocation Moves the empty tRNA to the E site, the peptide tRNA to the P site, and opens up the A site Repeat until a termination codon is encountered

All polypeptides at the time of synthesis begin with a _________________

Methionine

The protein is synthesized from its ____ terminus to its ___ terminus

N- C-

No Beta globin: Disease is more severe, and due to______________ (3)

Nonsense mutations, Frameshift mutations, and some splice junction mutations

Fate of proteins synthesized on the RER.

Proteins synthesized on ribosomes attached to the ER travel in vesicles to the cis face of the Golgi complex. After the mem-branes fuse, the proteins enter the Golgi complex. Structural features of the proteins deter-mine their fate. Some remain in the Golgi complex and some return to the RER. Others bud from the trans face of the Golgi complex in vesicles. These vesicles can become lysosomes or secretory vesicles, depending on their contents. Secretory proteins are released from the cell when secretory vesicles fuse with the cell membrane (exocytosis). Proteins with hydro-phobic regions embedded in the membrane of secretory vesicles become cell membrane proteins.

How does the cell ensure that 1 mRNA = 1 protein?

Reading frame is maintained

Recycling of elongation factors during translation

Recycling of eEF1A in eukaryotes. eEF1A contains a GTPase activity, which is activated upon binding to the ribosome. GTP is hydrolyzed, and eEF1A is released from the ribosome, binding to eEF1B(alpha). eEF1B(alpha) is a guanine nucleotide exchange factor and accelerates the substitution of GTP for the GDP on eEF1A. Once this occurs, eEF1A is ready for another round of translation. In prokaryotes, eEF1A cor-responds to EF-Tu, and the protein corresponding to eEF1B(alpha) is EF-Ts.

Sickle cell mutation

Sickle cell anemia is caused by a missense mutation. In each of the alleles for beta-globin. In the sickle cell gene, GTG replaces the nor-mal GAG. Thus, in the mRNA, the codon GUG replaces GAG and a valine residue replaces a glutamate residue in the protein. The amino acid change is indicated as E6V; the normal glutamate (E) at position 6 of the beta-chain has been replaced by a valine (V).

Why does the Met-tRNAi have to be in the P site at initiation?

The A site has to be available for the 2nd amino acid

Formation of aminoacyl-tRNA.

The amino acid is first activated by reacting with ATP. The amino acid is then transferred from the aminoacyl-AMP to tRNA.

What is a polysome?

The complex of mRNA and multiple ribosomes, each of which is producing a polypeptide chain. A single ribosome covers approximately 80 nucleotides of mRNA. Therefore, ribosomes are positioned on mRNA at intervals of approximately 100 nucleotides.

Is there tRNAs with anticodons that recognize the termination codons?

There are NO tRNAs with anticodons that recognize the termination codons

Three of the 64 possible codons terminate protein synthesis and are known as "stop" or nonsense codons, they are:_________.

UAA, UGA, UAG "U Are Annoying", "U Go Away", "U Are Gone"

_____________: each codon encodes only for one amino acid

Unambiguous

What determines which aminoacyl-tRNA will bind at the A site during elongation?

When Met-tRNAi (or a peptidyl-tRNA) is bound to the P site, the mRNA codon in the A site determines which aminoacyl-tRNA will bind to that site.

A tRNA that contains an amino acid attached covalently to its 3'-end is called an ________________ and is said to be "charged."

aminoacyl-tRNA

Amino acids are attached to their tRNAs by highly specific enzymes known as ___________________

aminoacyl-tRNA synthetases.

All amino acids are charged on appropriate tRNAs = ______________________

aminoacyl-tRNAs

When binding to the regulatory proteins prior to translation initiation, the mRNA has a secondary structure (contains stem loops), some of the initiation factors upon binding to the mRNA-protein complex have a ___________ activity which relieves the stem loop structures.

helicase

Signaling peptide recognized by SRP is usually a ______________ amino acid portion of the peptide

hydrophobic

Prokaryotes and eukaryotes have different mechanisms for translation ________________

initiation