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Gene Expression: Translation
Text authored by Dr. Peter J. Russell Slides authored by Dr. James R. Jabbur CHAPTER 6 Gene Expression: Translation
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Chemical Structure of Proteins
Protein Structure Chemical Structure of Proteins Proteins are built from amino acids held together by peptide bonds. The amino acids confer shape and properties to the protein One or more polypeptide chains may associate to form a protein complex. Each cell type has characteristic proteins that are associated with its function Amino acids are organic molecules with a chiral carbon center and 4 associated substituents All amino acids (with the exception of proline) have a common structure
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The a-carbon, which is a chiral center, is bonded to:
An amino group (NH2; physiologically it is NH+3) A Carboxyl group (COOH; physiologically it is COO-) A Hydrogen atom (H) and… an R group, which is different for each amino acid and confers distinctive properties for the amino acid. The R groups in an amino acid chain confer specific structural and functional properties to polypeptides Figure 6.2 Structures of the 20 naturally occurring amino acids, organized according to chemical type. Below each amino acid name are its three-letter and one-letter abbreviations.
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R GROUPS: Nonpolar Functional Groups Polar Functional Groups Acidic
Glycine (Gly or G) Alanine (Ala or A) Valine (Val or V) Leucine (Leu or L) Isoleucine (Ile or I) Methionine (Met or M) Phenylalanine (Phe or F) Trypotphan (Trp or W) Proline (Pro or P) Polar Functional Groups Serine (Ser or S) Threonine (Thr or T) Cysteine (Cys or C) Tyrosine (Tyr or Y) Asparagine (Asn or N) Glutamine (Gln or Q) Acidic Basic Electrically Charged (+ or -) Functional Groups Aspartic acid (Asp or D) Glutamic acid (Glu or E) Lysine (Lys or K) Arginine (Arg or R) Histidine (His or H)
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The N-terminal end defines the beginning of the polypeptide
Polypeptides are chains of amino acids joined by covalent peptide bonds (amide) A peptide bond forms between the carboxyl group of one amino acid, and the amino group of another Polypeptides are un-branched and have a free amino group at one end (the N terminus) and a carboxyl group at the other end (the C terminus) The N-terminal end defines the beginning of the polypeptide Figure 6.3 Peptide bond formation.
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Molecular Structure of Proteins
A functional protein consists of one or more polypeptides twisted, folded, and coiled into a unique three-dimensional shape (structure) Protein structure determines function! Antibody protein Protein from flu virus
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Four Levels of Protein Structure
Primary Structure Secondary Structure Tertiary Structure Quaternary Structure b pleated sheet +H3N Amino end Examples of amino acid subunits a helix Animation: Protein Structure Introduction
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Animation: Primary Protein Structure
The primary structure of a protein is its unique sequence of amino acids, like the order of letters in a long word Primary structure is determined by inherited genetic information encoded in DNA Animation: Primary Protein Structure
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+H3N Primary Structure Amino end Amino acid subunits 1 5 10 15 20 25
75 80 Figure 5.21 Levels of protein structure—primary structure 85 90 20 95 105 100 110 25 115 120 125 Carboxyl end
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Animation: Secondary Protein Structure
Secondary structure consists of coils and folds in the polypeptide chain The coils and folds of secondary structure result from hydrogen bonds between repeating constituents of the polypeptide backbone Typical secondary structures are a coil called an helix and a folded structure called a pleated sheet For the Cell Biology Video An Idealized Alpha Helix: No Sidechains, go to Animation and Video Files. For the Cell Biology Video An Idealized Alpha Helix, go to Animation and Video Files. For the Cell Biology Video An Idealized Beta Pleated Sheet Cartoon, go to Animation and Video Files. For the Cell Biology Video An Idealized Beta Pleated Sheet, go to Animation and Video Files. Animation: Secondary Protein Structure
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Secondary Structure b pleated sheet Examples of amino acid subunits
Figure 5.21 Levels of protein structure—secondary structure a helix
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Animation: Tertiary Protein Structure
Tertiary structure is determined by interactions between R groups, rather than interactions between backbone constituents These interactions between R groups include hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals interactions Strong covalent bonds called disulfide bridges may reinforce the protein’s structure Animation: Tertiary Protein Structure
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Hydrophobic interactions & van der Waals interactions Hydrogen bond
Polypeptide backbone Hydrogen bond Disulfide bridge Figure 5.21 Levels of protein structure—tertiary and quaternary structures Ionic bond
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Animation: Quaternary Protein Structure
Quaternary structure results when a protein consists of multiple polypeptide chains Tertiary Structure Quaternary Structure Animation: Quaternary Protein Structure
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Example: Collagen is a fibrous protein consisting of three polypeptides coiled like a rope
Example: Hemoglobin is a globular protein consisting of four polypeptides: two alpha and two beta chains Figure 5.21 Levels of protein structure—tertiary and quaternary structures
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Collagen Hemoglobin Polypeptide b Chains chain Iron Heme a Chains
Figure 5.21 Levels of protein structure—tertiary and quaternary structures Iron Heme a Chains Collagen Hemoglobin
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The Nature of the Genetic Code
Evidence for a Triplet Genetic Code Virulent wt+ bacteriophage T4 produces plaques on an infected lawn of Petri plated E. coli (roughly 200 progeny/bacterium) The rII mutant phage strain was generated by treating wt+ (r+) T4 phage with proflavin. Proflavin causes frameshift mutations by inserting or deleting base pairs of DNA; rII mutant phage do not grow in E. coli strain K12(l) Crick and colleagues reasoned the reversion of a deletion (a - mutation) could be caused by a nearby insertion (a + mutation) and vice versa. Revertants of rII to r+ were detected by plaques on E. coli K-12(l) [next slide] There were high levels of reversion only when combining genetically distinct rII mutants of the same type (either all - or all +) and only when in a combination of three (or multiple of three), indicating the genetic code is a triplet
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Example of a reversion of a deletion (frameshift) by a nearby addition (notice the successive repeat sequence TGC in wild-type DNA)
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Deciphering the genetic code
The relationship between codons and amino acids was determined using a cell-free, protein-synthesizing system from E. coli that included ribosomes and the required protein factors, along with tRNAs carrying radiolabeled amino acids To begin determining the genetic code, synthetic mRNAs were used in the cell-free translation system, and the resulting polypeptides were analyzed: When the mRNA contained one type of base, the results were consistent. For example, polyU (UUUUUUUUU) was responsible for a chain of 3 phenylalanines Using synthetic random copolymers of mRNA from 2 different nucleotides (i.e. A/G) produced 8 possible codons (2x2x2=8) Co-polymers with a known repeating sequence (i.e.; UCUCUCUCUCUC) produced polypeptides with alternating amino acids leu-ser-leu-ser However, this did not demonstrate which code was for which amino acid The ribosome-binding assay was another utilized approach: An in vitro translation system was generated, including: i. Ribosomes ii. tRNAs charged with their respective amino acids iii. A sequence-specific RNA trinucleotide The specific nucleotide sequences of the codons were determined this way
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Characteristics of the Genetic Code
It is a triplet code, non-overlapping and continuous 1 codon is the “start” sequence and encodes Methonine (AUG) 3 codons are the “stop” sequence to end translation (UGA, UAG, UAA) The genetic code is degenerate; no codon specifies more than one amino acid but 1 amino acid can be encoded several codons Codons must be read in the correct reading frame (correct groupings) in order for the specified polypeptide to be produced ORF’s and Wobble… Second mRNA base First mRNA base (5’ end of codon) Third mRNA base (3’ end of codon)
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An open reading frame (ORF) is a DNA sequence that contains a start codon but does not contain a stop codon in a given reading frame. For example, If a portion of a genome has been sequenced (5'-ATGTAGAATGGGTAAC-3'), ORFs can be located by examining each of the three possible reading frames on each strand. As an example, in this DNA sequence, two out of three possible reading frames are entirely open, meaning that they do not contain a stop codon: ...A TGT AGA ATG GGT AAC... ...AT GTA GAA TGG GTA AC... ...ATG TAG AAT GGG TAA C... Possible stop codons in DNA are "TGA", "TAA" and "TAG". Thus, the last reading frame in this example contains a stop codon (TAA), unlike the first two.
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Wobble occurs in the anticodon
Wobble occurs in the anticodon. The third 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 to recognize up to three different codons
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Translation: The Process of Protein Synthesis
Ribosomes translate the genetic message of mRNA into proteins. The mRNA is translated 5’ 3’, producing a corresponding N-terminal C-terminal polypeptide Amino acids are inserted into this polypeptide in the proper sequence due to: Specific binding of each amino acid to its tRNA Specific base-pairing between the mRNA and tRNA (codon/anticodon interaction) Animation: Protein Synthesis
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Transfer RNA (tRNA) A tRNA molecule consists of a single RNA strand that is only about 80 nucleotides long Flattened into one plane to reveal its base pairing, a tRNA molecule looks like a cloverleaf. But in reality, because of hydrogen bonding, tRNA actually twists and folds into a three-dimensional molecule which is roughly L-shaped In both prokaryotes and eukaryotes, their pre-tRNA molecules have end sequences removed and a CCA sequence added at the 3’ end
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…in addition, the contrasts
Prokaryotes Eukaryotes RNA pol transcribes everything RNA pol II only for tRNA tRNA genes are few in number tRNA genes are repeated Introns are found sometime Novel splicing mechanisms
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Adding an Amino Acid to tRNA
Aminoacyl-tRNA synthetase is the enzyme which attaches a specific amino acid to its specific tRNA The charging process, which is termed aminoacylation, produces a charged molecule of tRNA (aminoacyl-tRNA) This reaction is driven using energy from ATP hydrolysis There are 20 different aminoacyl-tRNA synthetase enzymes, one for each amino acid, and each recognizes the structure of the specific tRNA it charges The exact binding of a specific amino acid and its tRNA takes place via the following steps (next slide)…
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2. The amino acid specific tRNA is bound onto the enzyme.
The Loading process… Aminoacyl-tRNA synthetase (enzyme) Amino acid 1. The amino acid is bound onto the enzyme, requiring 2 ATP equivalents of energy from 1 ATP molecule. The amino acid is now in a ‘charged state.’ P P P Adenosine ATP P Adenosine tRNA P P 2. The amino acid specific tRNA is bound onto the enzyme. i Aminoacyl-tRNA synthetase P i P i Amino acid specific tRNA 3. The amino acid is LOADED onto the tRNA, creating a charged Aminoacyl-tRNA Figure 6.9 Charging of a tRNA molecule by aminoacyl-tRNA synthetase to produce an aminoacyl-tRNA (charged tRNA). P Adenosine AMP Computer model Aminoacyl-tRNA (“charged tRNA”)
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Ribosomes and Ribosomal RNA
Ribosomes facilitate the specific coupling of charged tRNA anticodons with mRNA codons during the process of protein synthesis Ribosomes in both bacteria and eukaryotes consist of two subunits of unequal size (large and small), each with at least one rRNA and many ribosomal proteins The rRNAs are important in the ribosome’s structure and function [why? catalysis?]
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Sites on the ribosome perform specific functions
The A site binds incoming aminoacyl-tRNAs The P site contains the tRNA carrying the growing polypeptide chain The E site allows the exit of the tRNA after donating its amino acid The A and P sites consist of regions of both subunits of the ribosome, while the E site is a region of the large subunit
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The Ribosome (a) Computer model of functioning ribosome
Growing polypeptide Exit tunnel The Ribosome tRNA molecules Large subunit E P A Small subunit 5’ mRNA 3’ (a) Computer model of functioning ribosome P site (Peptidyl-tRNA binding site) A site (Aminoacyl-tRNA binding site) E site (Exit site) E P A Large subunit mRNA binding site Small subunit (b) Schematic model showing binding sites Amino end Growing polypeptide Next amino acid to be added to polypeptide chain E tRNA mRNA 3’ 5’ Codons (c) Schematic model with mRNA and tRNA
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Ribosomal RNA genes “rRNA transcription units” encode rRNA on DNA
In typical prokaryotes, there are seven rRNA coding regions scattered in the chromosome Each rRNA transcription unit has a single promoter, ordering the genes 16S-23S-5S with non-rRNA spacer sequences The pre-rRNA transcript associates with ribosomal proteins and is cleaved by RNases to release the 3 rRNAs, which then associate with ribosomal proteins to form functional subunits Eukaryotes generally have many copies of rRNA genes The three rRNA genes with homology to prokaryotic rRNA genes are ordered18S-5.8S-28S with non-rRNA spacer sequences, and are repeated 100 to 1,000 times to form rDNA repeat units which are produced by RNA polymerase I The nucleolus produces ribosomal subunits by forming around each rDNA repeat unit, facilitating pre-rRNA cleavage and its assembly with 5S rRNA and ribosomal proteins The 5S rRNA gene copies are located elsewhere in the genome and are transcribed by RNA polymerase III
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Building a Polypeptide – Protein Synthesis
Protein synthesis is similar in prokaryotes and eukaryotes (significant differences will be addressed) There are three stages of translation (protein synthesis: Initiation Elongation Termination All three stages require protein “factors” that aid in the translation process
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Initiation of Translation
The initiation of translation requires: An mRNA transcript as a template An assembled ribosome A specific initiator tRNA Initiation factors (IF’s) which are proteins 1 GTP for energy …and Mg2+ (magnesium is a divalent cation and facilitates catalysis) Animation: Initiation of Translation
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kck Prokaryotic translation begins with binding of the 30S ribosomal subunit complex (IF-3, IF-3) to mRNA near the AUG codon IF-3 helps the 30S ribosome subunit bind to the mRNA An additional upstream sequence of 8 to 12 nucleotides (called the Shine-Dalgarno sequence or Ribosome Binding Site) is necessary to orient the ribosome to the correct reading frame This sequence is purine rich (i.e. AGGAG), is complementary to the 3’ end of the 16S rRNA and is necessary for ribosome binding
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Next, GTP bound IF-2 facilitates initiator tRNA binding to the AUG sequence on the mRNA which is bound to the 30S subunit P site IF-1 blocks the A site during initiation (why is this important?) The intitator amino acid used in prokaryotes is formylmethionine. It is carried by a specific tRNA All other methionines throughout the protein are not formylized In general, fMet is subsequently removed from the finished product Finally, the 50S ribosomal subunit binds the complex, GTP is hydrolyzed and the IF’s are released, forming a 70S initiation complex
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Differences in initiation between prokaryotes & eukaryotes
Initiation is more complex in eukaryotes The initiation factors are termed “eIF’s” The initiator methionine is not modified in eukaryotes but as in prokaryotes, it is attached to a special tRNA Ribosome binding involves the 5’ cap, rather than a Shine-Delgarno sequence eIF-4F is a multimeric complex, including the cap-binding protein (CBP) which binds to the 5’ mRNA cap Next, the 40S subunit complex (initiator Met-tRNA, several eIFs and GTP) binds the cap forming the initiator complex The initiator complex scans the mRNA for a Kozak sequence that includes the AUG start codon (the scanning model) With is start found, the ribosomes bind, eIF’s are displaced and the 80S initiation complex is formed The eukaryotic mRNA poly-A tail also interacts with the 5’ cap. The PolyA binding protein (PABP) binds to the mRNA poly-A, and also binds a protein in eIF-4F on the cap, circularizing the mRNA and stimulating translation
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Elongation of the Polypeptide Chain
The elongation of the polypeptide has 3 steps: The binding of an aminoacyl-tRNA to the ribosome The formation of a peptide bond (bonding), and… The translocation of the ribosome to the next codon Animation: Elongation of Translation
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Binding of an Aminoacyl-tRNA
fMet-tRNA is already 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 EF-Tu is then recycled (with some help from others and GTP)
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Formation of Peptide Bond
In the P site, the bond between the amino acid and its tRNA is cleaved 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. The 23S rRNA possesses the catalytic activity (antibiotics act here) The tRNA in the A site now has the growing polypeptide chain attached
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Translocation of the Aminoacyl-tRNA
(4.) The EF-G-GTP complex binds to the ribosome complex, GTP is hydrolyzed and the ribosome moves one codon Thus, the peptidyl-tRNA is transferred from the A site to the P site on the ribosome (5.) The empty tRNA at the E site is released. Binding of a charged tRNA in the A site is blocked until the spent tRNA is released (6.) 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...
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…a note on Polyribosomes
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). Polyribosomes enable a cell to make many copies of a polypeptide very quickly Figure 6.15 Diagram of a polysome—a number of ribosomes, each translating the same mRNA sequentially.
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Animation: Termination
Termination of Translation Termination is signaled by a stop codon (UAA, UAG, UGA) which has no corresponding tRNA Release factors (RF’s) assist the ribosome in recognizing the stop codon and terminating translation (2. & 3.) RF1 recognizes UAA and UAG; RF2 recognizes UAA and UGA. This RF also has peptidyl transferase activity (4.) RF3 stimulates termination via GTP hydrolysis (1 GTP used*) (5.-7.) RRF (ribosome recycling factor) binds the A site, EF-G translocates the ribosome, RRF then releases the last uncharged tRNA and EF-G releases RRF, facilitating ribosome dissociaton In eukaryotes eRF1 recognizes all 3 stop codons. eRF3 stimulates termination. Ribosome recycling occurs without an equivalent of RRF Animation: Termination
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Protein Sorting in the Cell
Eukaryotes must secrete proteins to act elsewhere in the body and move proteins into their intracellular compartments to work Two populations of ribosomes are evident in eukaryotic cells Cytosolic ribosomes synthesize proteins that function in the cytosol of the cell Endoplasmic reticulum bound ribosomes make proteins of the endomembrane system and proteins that are secreted from the cell Ribosomes are identical and can switch from free to bound Protein synthesis begins in the cytosol. Signal sequences on the nascent polypeptide direct them to their destinations: If the signal sequence is hyrophilic, translation continues to its termination in the cytosol If the sequence is hydrophobic, translation stalls and the entire complex is relocated to the endoplasmic reticulum. Here is where the magic happens…
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The Signal Hypothesis involves the NSS, SRP and SP
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