Lecture 3 – Translation of mRNA; Protein synthesis and

effects of mutations (chapter 13) – (13/9)

The genetic basis of protein synthesis

 proteins are the active participants in cell structure and function

 coding genes - genes that encode polypeptides (these are transcribed into messenger RNA,
mRNA)
 the main function of the genetic material is to encode the production of cellular proteins (in
the correct cell, at the proper time and in suitable amounts)

Overview of gene expression

The genetic code

 provides the ability of an mRNA to be translated into a specific sequence of amino acids
(polypeptide)

Special codons

 AUG (methionine) = START CODON

 AUG specifies additional methionines within the coding sequence
 UAA, UAG and UGA = TERMINATION, or STOP CODONS
 the code is degenerate
 more than 1 codon can specify the same amino acid (for example: GGU, GGC, GGA
and GGG all code for glysine, they are therfore synonymous)
 in most instances, the third base is the degenerate base (wobble base)
 the code is nearly universal = almost all organisms have the same codon usage
 only a few rare exceptions have been noted (mitochondrial genes)

Amino acids

 20 amino acids that may be found in polypeptides

 each contains a different side chain, or R group
Nonpolar aminoacids

 hydrophobic
 no polarity (they have equal numbers of carboxyl and amine groups - > neutral charge, no
charge on the “R” group)
 often buried within the interior of a folded protein

Polar aminoacids

 hydrophilic
 charge on the “R” grouo
 more likely to be on the surface of the protein

Levels of structures in proteins

 there are 4 levels of structures in proteins:

1. primary
2. secondary
3. tertiary
4. quaternary
 a protein’s primary structure is its amino acid sequence
 within the cell, the protein will not be found in a linear state (it will adapt a compact 3-D
structure)
 folding begins during translation
 the progression from the primary to the 3-D structure is dictated by the amino acid
sequence within the polypeptide
 the primary structure of a protein folds to form regular, repeating shapes known as
secondary structures
 2 types of secondary structures
1. α helix
2. β sheet
 depends on the primary aa sequence: amino acid propertie
 these are stabilized by the formation of hydrogen bonds
 the short regions of secondary structure in a protein fold into a three-dimensional tertiary
structure
 the final conformation of proteins that are composed of a single polypeptide
 structure determined by hydrophobic and ionic interactions as well as hydrogen
bonds and Van der Waals interactions
 proteins made up of two or more polypeptides have a quaternary structure - protein
complex (individual proteins are often called subunits)


Structure and function of transferRNA (tRNA)

Recognition between tRNA and mRNA

 during mRNA-tRNA recognition, the anticodon in tRNA binds to a complementary codon
in mRNA

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tRNAs share common structural features

 the secondary structure of tRNAs exhibits a kind of ‘cloverleaf” pattern

 it contains:
 three stem-loop structures; Variable region
 an acceptor stem and 3’ single strand region
 the actual three-dimensional or tertiary structure of a tRNA involves additional folding
 in addition to the normal A, U, G and C nucleotides, tRNAs commonly contain modified
nucleotides (more than 60 of these have been found)
Charging of tRNAs

 the enzymes that attach amino acids to tRNAs are known as aminoacyl-tRNA synthetases
(20 types, 1 for each amino acid)
 aminoacyl-tRNA synthetases catalyze a two-step reaction involving three different
molecules (amino acid, tRNA and ATP)
  the aminoacyl-tRNA synthetases are responsible for the “second genetic code”, which
ensures an aminoacyl-tRNA synthetase to recognize tRNAs
 the selection of the correct amino acid must be highly accurate or the
polypeptides may be nonfunctional
 error rate is less than one in every 100,000
 sequences throughout the tRNA, including but not limited to the anticodon, are
used as recognition sites
 many modified bases are used as markers

tRNAs and the Wobble Rule

 as mentioned earlier, the genetic code is degenerate (with the exception of serine, arginine
and leucine, this degeneracy always occurs at the codons third position)
 Wobble hypothesis, Francis Crick, 1966
 in the codon-anticodon recognition process, the first two positions pair strictly
according to the A – U /G – C rule
 however, the third position can actually “wobble” or move a bit: ”wobble base /
position” (thus tolerating certain types of mismatches)
Ribosome structure and assembly

 translation occurs on the surface of a large macromolecular complex termed the ribosome
 bacterial cells have one type of ribosome (in their cytoplasm)
 eukaryotic cells have 2 types of ribosomes:
1. one type is found in the cytoplasm
2. the other is found in organelles (mitochondria;chloroplasts)
 unless otherwise noted the term eukaryotic ribosome refers to the ribosomes in the
cytosol
 a ribosome is composed of structures called the large and small subunits
 each subunit is formed from the assembly of:
 proteins
 rRNA
Functional sites of ribosomes

 during bacterial translation, the mRNA lies on the surface of the 30S subunit
 As a polypeptide is being synthesized, it exits through a hole within the 50S subunit
 ribosomes contain 3 discrete sites:
1. Peptidyl site (P site)
2. Aminoacyl site (A site)
3. Exit site (E site)
 translation can be viewed as occurring in 3 stages:
1. initiation
2. elongation
3. termination
Prokaryotic translation initiation

 the mRNA, initiator tRNA, and ribosomal subunits associate to form an initiation complex
 this process requires 3 Initiation Factors (IF1, IF2, IF3)
 the initiator tRNA recognizes the start codon in mRNA
 In bacteria, this tRNA is designated tRNAfmet (it carries a methionine that has been
covalently modified to N-formylmethionine)
 the start codon is AUG, but in some cases GUG or UUG (in all 3 cases, the first amino
acid is N-formylmethionine)
 the binding of mRNA to the 30S subunit is also facilitated by a ribosomal-binding site or
Shine-Dalgarno sequence (complementary to a sequence in the 16S rRNA)
The translation initiation stage in eukaryotes

 in eukaryotes, the assembly of the initiation complex is similar to that in bacteria but
additional factors are required
 eukaryotic Initiation Factors – eIF
 the initiator tRNA is designated tRNAmet (it carries a methionine rather than a
formylmethionine)
 the start codon for eukaryotic translation is also AUG
 it is usually the first AUG after the 5’ Cap
 the consensus sequence for optimal start codon recognition is shown here

 these rules are called Kozak’s rules (after Marilyn Kozak)

 with that in mind, the start codon for eukaryotic translation is usually the first AUG after
the 5’ Cap!
 translational initiation in eukaryotes can be summarized as such:
 a number of initiation factors bind to the 5’ cap in mRNA
 these are joined by a complex consisting of the 40S subunit, tRNA met, and other
initiation factors
 the entire assembly moves along the mRNA ‘scanning’ for the right start codon
(usually the first AUG)
 Once it finds this AUG, the 40S subunit binds to it
 the 60S subunit joins
 this forms the 80S initiation complex
 (after the protein is synthesized, the first amino acid (aa) is removed! Final protein is 1
aa shorter than the number of aa codons in a mRNA!)

The translation elongation stage

 during this stage, the amino acids are added to the polypeptide chain, one at a time
 the addition of each amino acid occurs via a series of steps
 This process, though complex, can occur at a remarkable rate
 In bacteria  ~15-20 amino acids per second
 In eukaryotes  ~2-6 amino acids per second
  16S rRNA (a part of the 30S ribosomal subunit) plays a key role in codon-anticodon
recognition (It can detect an incorrect tRNA bound at the A site and it will prevent
elongation until the mis paired tRNA is released)

The translation termination stage

 the final stage occurs when a stop codon is reached in the mRNA
 in most species there are three stop or nonsense codons (UAG, UAA, UGA)
 these codons are not recognized by tRNAs, but by proteins called release factors
(indeed, the 3-D structure of release factors mimics that of tRNAs)
Messenger RNA reading frame

 each mRNA can basically be "read" in 3 "frames" / triplet register / triplet reading
frames, if you do not necessarily start with a start codon
 sequence of codons important: start codon indicates in which 'frame’ the mRNA
sequence should be read; from the first AUG codon you usually have the longest ORF
 ORF: ‘open reading frame’ That part of a RNA that can be translated into the protein:
from a START to a STOP codon
Mutations: base changes

Very harmful:
 deletion / insertion of one base
 deletion / insertion of two bases
 especially at the beginning of an ORF / gene
 if it is at the end of the gene, the 'shorter' protein or protein different at the C-
terminal end can still be (somewhat) active!
Usually less harmful:
 base substitution (another AA, but can also be a stop codon)
 deletion / insertion of three bases (loss of, or addition of, an extra amino acid (aa))

Protein sorting

Final destination of a protein:

 cytosol
 organe.
 membrane
 secreted