1. Gene Expression: Translation
Text authored by Dr. Peter J. Russell
Slides authored by Dr. James R. Jabbur
CHAPTER 6
Gene Expression: Translation

2. 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

3. 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.

4. 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)

5. 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.

6. 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

7. 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

8. 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

9. +H3N Primary Structure Amino end Amino acid subunits 1 5 10 15 20 2575 80
Figure 5.21 Levels of protein structure—primary structure 85 90 20 95
105
100
110 25
115
120
125
Carboxyl end

10. 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

11. Secondary Structure b pleated sheet Examples of amino acid subunits
Figure 5.21 Levels of protein structure—secondary structure
a helix

12. 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

13. 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

14. Animation: Quaternary Protein Structure
Quaternary structure results when a protein consists of multiple polypeptide chains
Tertiary Structure
Quaternary Structure
Animation: Quaternary Protein Structure

15. 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

16. 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

17. 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

18. Example of a reversion of a deletion (frameshift) by a nearby addition (notice the successive repeat sequence TGC in wild-type DNA)

19. 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

20. 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)

21. 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.

22. 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

23. 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

24. 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

25. …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

26. 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)…

27. 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”)

28. 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?]

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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

36. 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

37. 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

38. 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

39. 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)

40. 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

41. 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...

42. …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.

43. 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

44. 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…

45. The Signal Hypothesis involves the NSS, SRP and SP