台大農藝系遺傳學 601 20000 Chapter 5 slide 1 Proteins Chemical Structure of Proteins 1. Proteins are built from amino acids held together by peptide bonds. The.

台大農藝系遺傳學 601 20000 Chapter 5 slide 1

ProteinsChemical Structure of Proteins

1. Proteins are built from amino acids held together by peptide bonds. The amino acids confer shape and properties to the protein.

2. Two or more polypeptide chains may associate to form a protein complex. Each cell type has characteristic proteins that are associated with its function.

3. All amino acids (except proline) have a common structure (Figure 6.1).

a. The α-carbon is bonded to:

i. An amino group (NH2), which is usually charged at cellular pH (NH3

+).

ii. A carboxyl group (COOH), which is also usually charged at cellular pH (COO-).

iii. A hydrogen atom (H).

iv. An R group, which is different for each amino acid, and confers distinctive properties. The R groups in an amino acid chain give polypeptides their structural and functional properties.

台大農藝系遺傳學 601 20000 Chapter 5 slide 2Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 6.1 General structural formula for an amino acid


4. There are 20 amino acids used in biological proteins. They are divided into subgroups according to the properties of their R groups (acidic, basic, neutral and polar, or neutral and nonpolar) (Figure 6.2).


Fig. 6.2 Structures of the 20 naturally occurring amino acids organized according to

chemical type



chemical type (continued)



chemical type (continued)


5. Polypeptides are chains of amino acids joined by covalent peptide bonds. A peptide bond forms between the carboxyl group of one amino acid, and the amino group of another (Figure 6.3).

6. Polypeptides are unbranched, and have a free amino group at one end (the N terminus) and a carboxyl group at the other (the C terminus). The N-terminal end defines the beginning of the polypeptide.


Fig. 6.3 Mechanism for peptide bond formation between the carboxyl group of one

amino acid and the amino group of another amino acid


ProteinsMolecular Structure of Proteins1. Proteins have up to four levels of organization (Figure 6.4):

a. Primary structure is the amino acid sequence of the polypeptide. This is determined by the nucleotide sequence of the corresponding gene.

b. Secondary structure is folding and twisting of regions within a polypeptide, resulting from electrostatic attractions and/or hydrogen bonding. Common examples are α-helix and β-pleated sheet.

c. Tertiary structure is the three-dimensional shape of a single polypeptide chain, often called its conformation. Tertiary structure arises from interactions between R groups on the amino acids of the polypeptide, and thus relates to primary structure.

d. Quaternary structure occurs in multi-subunit proteins, as a result of the association of polypeptide chains. Hemoglobin is an example, with two 141-amino-acid a polypeptides, and two 146-amino-acid β polypeptides (each associated with a heme group).

2. More than amino acid sequence alone determines the folding of a polypeptide into a functional protein. Cell biology experiments show that proteins in the molecular chaperone family assist other proteins in folding.


Fig. 6.4 Four levels of protein structure


The Nature of the Genetic Code

1. How many nucleotides are needed to specify one amino acid? A one-letter code could specify four amino acids; two-letters specify 16 (4 X 4). To accommodate 20, at least three letters are needed.


Fig. 6.7 The genetic code


Characteristics of the Genetic Code1. Characteristics of the genetic code:

a. It is a triplet code. Each three-nucleotide codon in the mRNA specifies 1 amino in the polypeptide.

b. It is comma free. The mRNA is read continuously, three bases at a time, without skipping any bases.

c. It is non-overlapping. Each nucleotide is part of only one codon, and is read only once during translation.

d. It is almost universal. In nearly all organisms studied, most codons have the same amino acid meaning. Examples of minor code differences include the protozoan Tetrahymena and mitochondria of some organisms.

e. It is degenerate. Of 20 amino acids, 18 are encoded by more than one codon. Met (AUG) and Trp (UGG) are the exceptions; all other amino acids correspond to a set of two or more codons. Codon sets often show a pattern in their sequences; variation at the third position is most common (Figure 6.8).

f. The code has start and stop signals. AUG is the usual start signal for protein synthesis. Stop signals are codons with no corresponding tRNA, the nonsense or chain-terminating codons. There are generally three stop codons: UAG (amber), UAA (ochre) and UGA (opal).

g. Wobble occurs in the anticodon. The 3rd base in the codon is able to base-pair less specifically, because it is less constrained three-dimensionally. It wobbles, allowing a tRNA with base modification of its anticodon (e.g., the purine inosine) to recognize up to three different codons (Figure 6.8).


Fig. 6.8 Example of base-pairing wobble


Translation: The Process of Protein Synthesis

1. Ribosomes translate the genetic message of mRNA into proteins.

2. The mRNA is translated 5’3’, producing a corresponding N-terminal C-terminal polypeptide.

3. Amino acids bound to tRNAs are inserted in the proper sequence due to:

a. Specific binding of each amino acid to its tRNA.

b. Specific base pairing between the mRNA codon and tRNA anticodon.


The mRNA Codon Recognizes the tRNA Anticodon

1. tRNA.Cys normally carries the amino acid cysteine. Ehrenstein, Weisblum and Benzer attached cysteine to tRNA.Cys (making Cys-tRNA.Cys), and then chemically altered it to alanine (making Ala-tRNA.Cys).

2. When used for in vitro synthesis of hemoglobin, the tRNA inserted alanine at sites where cysteine was expected.

3. The concluded that the specificity of codon recognition lies in the tRNA molecule, and not in the amino acid it carries.


Charging tRNA (Adding amino acid to tRNA)1. Aminoacyl-tRNA synthetase attaches amino acids to their specific

tRNA molecules. The charging process (aminoacylation) produces a charged tRNA (aminoacyl-tRNA), using energy from ATP hydrolysis.

2. There are 20 different aminoacyl-tRNA synthetase enzymes, one for each amino acid. Some of these enzymes recognize tRNAs by their anticodon regions, and others by sequences elsewhere in the tRNA.

3. The amino acid and ATP bind to the specific aminoacyl-tRNA synthetase enzyme. ATP loses two phosphates and the resulting AMP is bound to the amino acid, forming aminoacyl-AMP (Figure 6.9).

4. The tRNA binds to the enzyme, and the amino acid is transferred onto it, displacing the AMP. The aminoacyl-tRNA is released from the enzyme.

5. The amino acid is now covalently attached by its carboxyl group to the 3’r end of the tRNA. Every tRNA has a 3’r adenine, and the amino acid is attached to the 3’r–OH or 2’r–OH of this nucleotide.(Figure 6.10).


Initiation of TranslationAnimation: Initiation of Translation

1. Protein synthesis is similar in prokaryotes and eukaryotes. Some significant differences do occur, and are noted below.

2. In both it is divided into three stages:

a. Initiation.

b. Elongation.

c. Termination.

3. Initiation of translation requires:

a. An mRNA.

b. A ribosome.

c. A specific initiator tRNA.

d. Initiation factors.

e. Mg2+ (magnesium ions).


4. Prokaryotic translation begins with binding of the 30S ribosomal subunit to mRNA near the AUG codon (Figure 6.11). The 30S comes to the mRNA bound to:

a. All three initiation factors, IF1, IF2 and IF3.

b. GTP.

c. Mg2+.

5. Ribosome binding to mRNA requires more than the AUG:

a. RNase protection experiments have shown that the ribosome binds at a ribosome-binding site, where it is oriented to the correct reading frame for protein synthesis (Figure 6.13)

b. The AUG is clearly identified in these studies.

c. An additional sequence 8–12 nucleotides upstream from the AUG is commonly involved. Discovered by Shine and Dalgarno, these purine-rich sequences (e.g., AGGAGG) are complementary to the 3’r end of the 16S rRNA (Figure 6.12)

d. Complementarity between the Shine-Dalgarno sequence and the 3’r end of 16S rRNA appears to be important in ribosome binding to the mRNA


Fig. 6.11 Initiation of protein synthesis in prokaryotes


Fig. 6.12 Sequences involved in the binding of ribosomes to the mRNA in the

initiation of protein synthesis in prokaryotes

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.


6. Next, the initiator tRNA binds the AUG to which the 30S subunit is bound. AUG universally encodes methionine. Newly made proteins begin with Met, which is often subsequently removed.

a. Initiator methionine in prokaryotes is formylmethionine (fMet). It is carried by a specific tRNA (with the anticodon 5’r-CAU-3’r).

b. The tRNA first binds a methionine, and then transformylase attaches a formyl group to the methionine, making fMet-tRNA.fMET (a charged initiator tRNA).

c. Methionines at sites other than the beginning of a polypeptide are inserted by tRNA.Met (a different tRNA), which is charged by the same aminoacyl-tRNA synthetase as tRNA.fMet.

7. When Met-tRNA.fMet binds the 30S-mRNA complex, IF3 is released and the 50S ribosomal subunit binds the complex. GTP is hydrolysed, and IF1 and IF2 are relased. The result is a 70S initiation complex consisting of (Figure 6.14):

a. mRNA.

b. 70S ribosome (30S and 50S subunits) with a vacant A site.

c. fMet-tRNA in the ribosome’s P site.


8. The main differences in eukaryotic translation are:

a. Initiator methionine is not modified. As in prokaryotes, it is attached to a special tRNA.

b. Ribosome binding involves the 5’r cap, rather than a Shine-Dalgarno sequence.

i. Eukaryotic initiator factor (eIF-4F) is a multimer of proteins, including the cap binding protein (CBP), binds the 5’r mRNA cap.

ii. Then the 40S subunit, complexed with initiator Met-tRNA, several eIFs and GTP, binds the cap complex, along with other eIFs.

iii. The initiator complex scans the mRNA for a Kozak sequence that includes the AUG start codon. This is usually the 1st AUG in the transcript.

iv. When the start codon is located, 40S binds, and then 60S binds, displacing the eIFs and creating the 80S initiation complex with initiator Met-tRNA in the ribosome’s P site.

c. The eukaryotic mRNA’s 3’r poly(A) tail also interacts with the 5’r cap. Poly(A) binding protein (PABP) binds the poly(A), and also binds a protein in eIF-4F on the cap, circularizing the mRNA and stimulating translation.


Elongation of the Polypeptide Chain

Animation: Elongation of the Polypeptide Chain

1. Elongation of the amino acid chain has three steps (Figure 6.13):

a. Binding of aminoacyl-tRNA to the ribosome.

b.Formation of a peptide bond.

c. Translocation of the ribosome to the next codon.


Fig. 6.13 Elongation stage of translation in prokaryotes


Binding of Aminoacyl-tRNA

1. Protein synthesis begins with fMet-tRNA in the P site of the ribosome. The next charged tRNA approaches the ribosome bound to EF-Tu-GTP. When the charged tRNA hydrogen bonds with the codon in the ribosome’s A site, hydrolysis of GTP releases EF-Tu-GDP.

2. EF-Tu is recycled with assistance from EF-Ts, which removes the GDP and replaces it with GTP, preparing EF-Tu-GTP to escort another aminoacyl tRNA to the ribosome.


Peptide Bond Formation

1. The two aminoacyl-tRNAs are positioned by the ribosome for peptide bond formation, which occurs in two steps:(Fig. 6.14)

a. In the P site, the bond between the amino acid and its tRNA is cleaved.

b. Peptidyl transferase forms a peptide bond between the now-free amino acid in the P site and the amino acid attached to the tRNA in the A site. Experiments indicate that the 23S rRNA is most likely the catalyst for peptide bond formation.

c. The tRNA in the A site now has the growing polypeptide chain attached to it.


Fig. 6.14 The formation of a peptide bond between the first two amino acids of a

polypeptide chain is catalyzed on the ribosome by peptidyl transferase



Translocation1. The ribosome now advances one codon along the mRNA. EF-G is used in

translocation in prokaryotes. EF-G-GTP binds the ribosome, GTP is hydrolyzed and the ribosome moves 1 codon while the uncharged tRNA leaves the P site. Eukaryotes use a similar process, with a factor called eEF-2.

2. Release of the uncharged tRNA involves the 50S ribosomal E (for Exit) site. Binding of a charged tRNA in the A site is blocked until the spent tRNA is released from the E site.

3. During translocation the peptidyl-tRNA remains attached to its codon, but is transferred from the ribosomal A site to the P site by an unknown mechanism.

4. The vacant A site now contains a new codon, and an aminoacyl-tRNA with the correct anticodon can enter and bind. The process repeats until a stop codon is reached.

5. Elongation and translocation are similar in eukaryotes, except for differences in number and type of elongation factors and the exact sequence of events.

6. In both prokaryotes and eukaryotes, simultaneous translation occurs. New ribosomes may initiate as soon as the previous ribosome has moved away from the initiation site, creating a polyribosome (polysome); an average mRNA might have 8-10 ribosomes (Figure 6.15).


Fig. 6.15 Diagram of a polysome, a number of ribosomes each translating the same

mRNA sequentially



Termination of TranslationAnimation: Translation Termination

1. Termination is signaled by a stop codon (UAA, UAG, UGA), which has no corresponding tRNA (Figure 6.16).

2. Release factors (RF) assist the ribosome in recognizing the stop codon and terminating translation.

a. In E. coli:

i. RF1 recognizes UAA and UAG.

ii. RF2 recognizes UAA and UGA.

iii. RF3 stimulates termination.

b. In eukaryotes, there is only one termination factor, eRF.

3. Termination events triggered by release factors are:

a. Peptidyl transferase releases the polypeptide from the tRNA in the ribosomal P site.

b. The tRNA is released from the ribosome.

c. The two ribosomal subunits and RF dissociate from the mRNA.

d. The initiator amino acid (fMet or Met) is usually cleaved from the polypeptide.


Fig. 6.16 Termination of translation



Protein Sorting in the Cell1. Localization of the new protein results from signal (leader) sequences in the

polypeptide.

2. In eukaryotes, proteins synthesized on the rough ER (endoplasmic reticulum) are glycosylated and then transported in vesicles to the Golgi apparatus. The Golgi sorts proteins based on their signals, and sends them to their destinations.

a. The required signal sequence for a protein to enter the ER is 15–30 N-terminal amino acids.

b. As the signal sequence is produced by translation, it is bound by a signal recognition particle (SRP) composed of RNA and protein.(Fig. 6.17)

c. The SRP suspends translation until the complex (containing nascent protein, ribosome, mRNA and SRP) binds a docking protein on the ER membrane.

d. When the complex binds the docking protein, the signal sequence is inserted into the membrane, SRP is released, and translation resumes. The growing polypeptide is inserted through the membrane into the ER, an example of cotranslational transport.

e. In the ER cisternal space, the signal sequence is removed by signal peptidase and the protein is usually glycosylated.

f. Proteins destined for other organelles are translated completely, and then specific amino acid sequences direct their transport into the appropriate organelle.


Fig. 6.17x Movement of secretory proteins through the cell membrane system



Fig. 6.17 Model for the translocation of proteins into the endoplasmic reticulum in

eukaryotes


台大農藝系 遺傳學 601 20000 Chapter 5 slide 1 Proteins Chemical Structure of Proteins 1. Proteins are built from amino acids held together by peptide bonds. The.

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台大農藝系遺傳學 601 20000 Chapter 5 slide 1 Proteins Chemical Structure of Proteins 1. Proteins are built from amino acids held together by peptide bonds. The.