Unit Six

Outline
The Structure and Function of Proteins
Translation: Protein synthesis
The translational machinery
o The ribosome
o mRNA
o The adaptor, tRNA
o Aminoacyl-tRNA synthetase
Stages of prokaryotic translation
o Initiation, Elongation, & Termination
Stages of eukaryotic translation
o Initiation, Elongation, & Termination
Post translational processing
 The Structure and Function of Proteins
 Proteins are central to all living processes. Many proteins are enzymes,
others are structural components, providing scaffolding and support for
membranes, filaments, bone, and hair. Some proteins help transport
substances; others have a regulatory, communication, or defense function.
 All proteins are composed of amino acids, linked end to end. Twenty
common amino acids are found in proteins. (Other amino acids sometimes
found in proteins are modified forms of the common amino acids.)
 The 20 common amino acids are similar in structure: each consists of a
central carbon atom bonded to an amino group, a hydrogen atom, a
carboxyl group, and an R (radical) group that differs for each amino acid.
 The amino acids in proteins are joined together by peptide bonds (Figure) to
form polypeptide chains, and a protein consists of one or more polypeptide
chains.
 Like nucleic acids, polypeptides have polarity, with one end having a free
amino group (NH3+) and the other end possessing a free carboxyl group
(COO−).
 Some proteins consist of only a few amino acids, whereas others may have
thousands.
 The Structure and Function of Proteins
 Protein structure Like that of nucleic acids, the molecular
structure of proteins has several levels of organization.
 The primary structure of a protein is its sequence of amino
acids (Figure).
 Through interactions between neighboring amino acids, a
polypeptide chain folds and twists into a secondary structure
(Figure); two common secondary structures found in proteins
are the beta (β) pleated sheet and the alpha (α) helix.
 Secondary structures interact and fold further to form a
tertiary structure (Figure), which is the overall, three-
dimensional shape of the protein.
 The secondary and tertiary structures of a protein are largely
determined by the primary structure—the amino acid
sequence—of the protein.
 Finally, some proteins consist of two or more polypeptide
chains that associate to produce a quaternary structure.
 Translation: Protein synthesis
 The nucleotides in the mRNA is used to
generate the linear sequences of amino acids in
protein in a process known as translation.
 Transcription is simple to understand as a
means of information transfer: since DNA & RNA
are chemically and structurally similar
 In contrast, the conversion of the information in
RNA into protein represents a translation of the
information into another language.
 The machinery responsible for translating the
language of mRNAs into the language of
proteins is composed of four primary
components:
 mRNA, tRNA, Aminoacyl tRNA synthetase and
Ribosome.
 The ribosome
 The ribosome is a place
where protein synthesis is
performed.
 catalytic machine made from
• more than 50 different
proteins (the ribosomal
proteins) &
• several RNA molecules, the
ribosomal RNAs (rRNAs).
 Eukaryotic & bacterial ribosomes
have similar structures &
functions
 composed of one large & one
small subunit that fit together to
form a complete ribosome
 they are assembled at the nucleolus
 ribosomal subunits are then exported to the cytoplasm, where they join together
 Small subunit
• provides the framework on which the tRNAs
are accurately matched to the codons of the
mRNA,
while the large subunit
catalyzes the formation of the peptide bonds
that link the amino acids together
 the two subunits are separate when not
actively synthesizing proteins
 join together on an mRNA molecule, usually
near its 5ʹ end, to initiate protein synthesis
 Large subunit contains the peptidyl transfer
center for peptide bond formation
 small subunit contains the decoding center
the charged tRNAs read or ‘decode’ the
codon units of the mRNA
 synthesis of most protein molecules takes b/n 20 sec. &
several minutes.
 multiple initiations to take place on each mRNA molecule
being translated
 the mRNA molecules being translated are usually found in
the form of polyribosomes (or polysomes)
 It is a large cytoplasmic assemblies made up of several
ribosomes
 This allow the cell to make many more protein molecules
in a given time
A ribosome contains four binding sites: 1 is for the mRNA
& 3 (A site, P site, & E site) are for tRNAs.
• Aminoacyl site (A site or entry site): accepts tRNA
charged with a single amino acid;
• Peptidyl site (P site): holds a tRNA linked to a peptide
chain and
• Exit site (E site): is occupied by a tRNA that is about to
be released
A tRNA molecule is held tightly at the A and P sites only
if its anticodon forms base pairs with a complementary
codon
 mRNA
• The genetic information encoded on the mRNA
• mRNA possess information for protein synthesis in the form of nucleotide
triplets called Codons
• The coding region which is composed of continuous, non-overlapping
nucleotide sequence free from stop codons (& thus with the potential to code for
protein) called Open Reading Frame (ORF).
• The No of ORFs vary in prokaryotes & eukaryotes
• bacterial ribosome can readily assemble directly on a start codon that lies in the
interior of an mRNA molecule, bacterial mRNAs are often polycistronic—that is,
they encode several different proteins
 The Ribosome Binding Site
 eukaryotic mRNA recruit the small
ribosomal subunit to the 5ʹ end of an
mRNA molecule, which is recognized by its
5ʹ cap.
 Once the mRNA is bound, the ribosome
moves in a 5’-3’ direction until it encounters
a start codon – This is called Scanning.
 Bacterial mRNAs have no 5ʹ caps to signal
the ribosome where to begin searching for
the start of translation
 Instead, each bacterial mRNA contains a
specific ribosome-binding site (called
Shine–Dalgarno sequence) that is located
a few nucleotides upstream of the AUG
(start codon)
 with the consensus 5ʹ-AGGAGGU-3ʹ, forms
base pairs with the 16S rRNA of the small
ribosomal subunit to position the initiating
AUG codon in the ribosome
 The adaptor, tRNA
• Eukaryotic tRNAs are synthesized by RNA pol. III
• Both bacterial & eukaryotic tRNAs are typically
synthesized as larger precursor tRNAs, which are
then trimmed to produce the mature tRNA
• codons in an mRNA molecule do not directly recognize
the amino acids they specify.
• Rather, the translation of mRNA into protein depends
on adaptor molecules that can recognize & bind both
to the codon &, at another site on their surface, to the
amino acid.
• The adaptor molecule for translation is the tRNA.
• Each type of tRNA carries a specific amino acid (at
the 3' terminus) & possesses a three-base sequence
called the anticodon
⁑⁑Therefore, the tRNA serves as the adaptor
between codons in the mRNA and amino acids
 Contd.
 tRNAs contain some unusual bases (chemical
modification after synthesis). For example, the bases
denoted ψ (pseudouridine) & D (dihydrouridine) are
derived from uracil
 have a relatively short size (70- 80 nucleotides).
 There are many types of tRNAs, but each is attached
to a specific amino acid & each recognizes a particular
codon, or codons.
 all tRNAs have a 2D cloverleaf structure while the 3D
structure looks like an L shape. The cloverleaf has four
major arms, each consisting of a stem and a loop.
 the acceptor, the TψC, the anticodon, & the DHU arms.
 The acceptor arm: has no loop but contains both the
5`and 3` ends of the tRNA molecule. It is the arm where
the amino acid is attached to its 3’ end. All tRNAs have
the same sequence, CCA, at this 3`end.
 Contd.
 The TψC arm: is named for the bases of three
nucleotides in the loop of this arm: thymine (T), pseudo
uracil (ψ), and cytosine (C).
 The anticodon arm: lies at the bottom of the tRNA. Three
nucleotides at the end of this arm make up the anticodon,
which pairs with the corresponding codon on mRNA to
ensure that the amino acids link in the correct order.
 The DHU arm: is so named because it often contains the
modified base dihydrouridine.
 The sequence of nucleotides in the mRNA molecule is
read in consecutive groups of 3 & RNA is a linear polymer
of 4 different nucleotides
 The Degeneracy of the Code
 One amino acid is encoded by three consecutive nucleotides in mRNA,
and each nucleotide can have one of four possible bases (A, G, C, and U) at
each nucleotide position, thus permitting 43 = 64 possible codons.
 Three of these codons are stop codons, specifying the end of translation.
Thus, 61 codons, called sense codons, encode amino acids. Because there
are 61 sense codons and only 20 different amino acids commonly found in
proteins, the code contains more information than is needed to specify the
amino acids and is said to be a degenerate code.
 This expression does not mean that the genetic code is depraved; degenerate
is a term that Francis Crick borrowed from quantum physics, where it
describes multiple physical states that have equivalent meaning.
 The degeneracy of the genetic code means that amino acids may be
specified by more than one codon. Only tryptophan and methionine are
encoded by a single codon.
 Other amino acids are specified by two codons, and some, such as leucine,
are specified by six different codons.
 Codons that specify the same amino acid are said to be synonymous, just
as synonymous words are different words that have the same meaning.
 The Degeneracy of the Code contd.
 tRNAs serve as adapter molecules, binding particular amino acids and delivering them
to a ribosome, where the amino acids are then assembled into polypeptide chains.
 Each type of tRNA attaches to a single type of amino acid. The cells of most
organisms possess from about 30 to 50 different tRNAs, and yet there are only 20
different amino acids in proteins. Thus, some amino acids are carried by more than
one tRNA. Different tRNAs that accept the same amino acid but have different
anticodons are called isoaccepting tRNAs.
 Even though some amino acids have multiple (isoaccepting) tRNAs, there are still
more codons than anticodons, because different codons can sometimes pair with
the same anticodon through flexibility in base pairing at the third position of the
codon. For example, alanine is encoded by the codons GCU, GCC, GCA, and
GCG, all of which begin with GC.
 When the codon on the mRNA and the anticodon of the tRNA join (Figure), the first
(5′) base of the codon pairs with the third (3′) base of the anticodon, strictly
according to Watson-and-Crick rules: A with U; C with G.
 Next, the middle bases of codon and anticodon pair, also strictly following the
Watson-and-Crick rules. After these pairs have hydrogen bonded, the third bases
pair weakly: there may be flexibility, or wobble, in their pairing.
 The Degeneracy of the Code contd.
 In 1966, Francis Crick developed the wobble hypothesis, which proposed
that some nonstandard pairings of bases could take place at the third
position of a codon.
 For example, a G in the anticodon may pair with either a C or a U in the third
position of the codon (Table).
 The important thing to remember about wobble is that it allows some
tRNAs to pair with more than one codon on an mRNA; thus from 30 to 50
tRNAs can pair with 61 sense codons. Some codons are synonymous
through wobble.
 redundancy implies
o that there is more than one tRNA for many of the amino acids
o that some tRNA molecules can base-pair with more than one codon.
 some tRNAs require accurate base-pairing only at the first two positions
of the codon & can tolerate a mismatch (or wobble) at the third position.
 This wobble base-pairing explains why so many of the alternative codons
for an amino acid differ only in their third nucleotide
 The first 2 nucleotides strictly follow base pair complementarity
 But the third base pairs weakly & there might be flexibility in pairing or
wobbles in its pairing
 The Reading Frame and Initiation Codons
 Findings from early studies of the genetic code indicated that the code
is generally nonoverlapping.
 An overlapping code is one in which a single nucleotide may be
included in more than one codon (see the figure)
 Usually, however, each nucleotide is part of a single codon. A few
overlapping genes are found in viruses, but codons within the same
gene do not overlap, and the genetic code is generally considered to
be nonoverlapping.
 For any sequence of nucleotides, there are three potential sets of
codons—three ways in which the sequence can be read in groups of
three.
 Each different way of reading the sequence is called a reading frame, and any sequence of nucleotides has three
potential reading frames. The three reading frames have completely different sets of codons and will therefore
specify proteins with entirely different amino acid sequences. Thus, it is essential for the translational machinery
to use the correct reading frame. How is the correct reading frame established?
The reading frame is set by the initiation codon, which is the first codon of the mRNA to specify an amino acid.
 After the initiation codon, the other codons are read as successive groups of three nucleotides.
 No bases are skipped between the codons; so there are no punctuation marks to separate the codons.
 The Reading Frame and Initiation Codons
 The initiation codon is usually AUG, although GUG and UUG are used on rare occasions.
 The initiation codon is not just a sequence that marks the beginning of translation; it
specifies an amino acid.
 In bacterial cells, AUG encodes a modified type of methionine, N-formylmethionine; all
proteins in bacteria initially begin with this amino acid, but the formyl group (or, in some
cases, the entire amino acid) may be removed after the protein has been synthesized.
 When the codon AUG is at an internal position in a gene, it encodes unformylated
methionine.
 In archaeal and eukaryotic cells, AUG specifies unformylated methionine both at the initiation
position and at internal positions.
Termination Codons
 Three codons—UAA, UAG, and UGA—do not encode amino acids. These codons signal the
end of the protein in both bacterial and eukaryotic cells and are called stop codons,
termination codons, or nonsense codons. No tRNA molecules have anticodons that pair with
termination codons.
 The Universality of the Code

 For many years the genetic code was assumed to

be universal, meaning that each codon specifies
the same amino acid in all organisms. We now
know that the genetic code is almost, but not
completely, universal; a few exceptions have been
found.
 Most of these exceptions are termination codons,
but there are a few cases in which one sense
codon substitutes for another.
 Most exceptions are found in mitochondrial
genes; a few nonuniversal codons have also
been detected in the nuclear genes of protozoans
and in bacterial DNA.
 The Binding of Amino Acids to Transfer RNAs
 The first stage of translation is the binding of tRNA molecules to their appropriate amino acids, called tRNA
charging. Each tRNA is specific for a particular amino acid.
 All tRNAs have the sequence CCA at the 3′ end, and the carboxyl group (COO–) of the amino acid is attached to
the adenine nucleotide at the 3′ end of the tRNA (Figure).
 If each tRNA is specific for a particular amino acid but all amino acids are attached to the same nucleotide (A) at
the 3′ end of a tRNA, how does a tRNA link up with its appropriate amino acid?
 The key to specificity between an amino acid and its tRNA is a set of enzymes called aminoacyl-tRNA synthe
tases. A cell has 20 different aminoacyl-tRNA synthetases, one for each of the 20 amino acids.
Each synthetase recognizes a particular amino acid, as well as all the tRNAs that accept that amino acid.
 Aminoacyl tRNA synthetase.
 Recognition & attachment of the correct amino acid depends on enzymes
called aminoacyl-tRNA synthetases, which covalently couple each
amino acid to its appropriate set of tRNA molecules.
 different synthetase enzyme for each amino acid (one attaches glycine to
all tRNAs that recognize codons for glycine, another attaches alanine to
all tRNAs that recognize codons for alanine, so on).
 Many bacteria, however, have fewer than 20 synthetases, & the same
synthetase enzyme is responsible for coupling more than one amino
acid to the appropriate tRNAs.
 The attachment of amino acid to tRNA at the 3’ end of the acceptor arm is
called charging of tRNA.
 the carboxyl group of an amino acid is attached to the 2’ or 3’ OH group at
the 3’ end of the tRNA
 the attachment of a particular amino acid to a particular tRNA is specified
by aminoacyl-tRNA synthetase.
 Recognition of the appropriate amino acid by a synthetase is based on the
different sizes, charges, and R groups of the amino acids.
 The recognition of tRNAs by a synthetase depends on the differing
nucleotide sequences of the tRNAs.
 Stages of prokaryotic translation
• the translation of a genetic message into the primary sequence of a polypeptide
can be divided into three phases: initiation, elongation and termination.
Initiation
 The site at which protein synthesis begins on the mRNA is especially crucial. The
initiation step is also important
 The translation of an mRNA begins with the codon AUG, & a special tRNA is
required to start translation.
 This initiator tRNA always carries the amino acid methionine (in bacteria,
formylmethionine is used), with the result that all newly made proteins have
methionine as the first amino acid at their N-terminus, (this methionine is usually
removed later).
 Translation is initiated when the small ribosomal subunit (30S) binds to the mRNA
and the initiator tRNA–formylmethionine (fmet-tRNAfmet) complex along with several
initiation proteins called IF1, IF2, and IF3
 Initiation (contd.)
• During initiation, the nucleotides in the Shine-
Dalgarno sequence of the mRNA pair with their
complementary nucleotides in the 16S rRNA of the
ribosomes to position the initiating AUG codon in
the ribosome.
• Then, the fmet-tRNAfmet binds to the initiation codon,
AUG. The methionine is formylated at the N-terminus
of the amino acid to allow chain elongation only from
the C-end (thus elongation is only from N-C end).
• The fMet-tRNAfMet must always attach to the P site.
The attachment is facilitated by initiation factor
2(IF2), which forms a complex with GTP.
• IF1 prevents the fMet-tRNAfMet from incorrectly
binding to the A site.
 the 30S ribosome; the mRNA; the fMet-tRNAfMet,
GTP; and IF-3, IF-2, and IF-1 aligned with the start
codon is known as the 30S initiation complex
 When the large subunit has joined the initiation
complex, it is called the 70S initiation complex.
 Elongation
• It is the stage where amino acids are joined to
create a polypeptide chain. Elongation
requires the 70S complex formed during
initiation, tRNAs charged with their amino
acids; several elongation factors & GTP
• Two elongation factors, each hydrolyzing GTP
to GDP & undergoing conformational
changes in the process (EF-Tu and EF-G in
bacteria, & EF1 & EF2 in eukaryotes).
• After initiation, the fmet-tRNAfmet is positioned
over the AUG start codon on the P site while
the A site is free with the first codon ready for
the next incoming charged tRNA.
• Elongation takes place in 3 stages
1. Placement of the charged tRNA in the A site
• Elongation factor EF-Tu-GTP binds to the next
aminoacylated tRNA & guides it to the A site.
 Elongation (contd.)
• The anticodon of the tRNA pairs with the
codon on the mRNA
• The EF-TU- GTP rotates the charged tRNA on
the A site in to position for peptide bond
formation utilizing energy (dissociation of GTP)
• Then, EF-Tu-GDP dissociates from the ribosome
& is recycled in to its active form by EF-Ts.
2. Peptide bond formation
• An enzyme peptidyl transferase catalyzes
the reaction that transfers the amino acids
from the tRNA (fMet) on the P site to the tRNA
on the A site
• A peptide bond is therefore formed b/n fMet &
the aa on the tRNA (has occupied the A site)
• The catalytic activity is carried out by a rRNA
found in the large subunit of the ribosome
(ribozyme).
Peptide Bond Formation
Carboxyl Amino group
group

26
 Elongation (contd.)
3. Translocation
• For continued synthesis, the next codon and
the A site should be empty & thus available for
the next charged tRNA to be admitted.
• Thus, the 30S ribosome moves one codon
further along the mRNA in the 5’-3’ direction
(facilitated by EF-G (translocase) & GTP).
• This process is called translocation & it
simultaneously transfers the discharged tRNA
(tRNAfmet) from P site to E site; the tRNA
charged with the dipeptide from the A site to
the P site & leaves the A site empty for the
next codon to be decoded & allow the binding
of the next tRNA.
• When the discharged tRNA enters the E site, it
dissociates from the ribosome.
• Sequential formation of polypeptide continues
accordingly.
 Termination
• Protein synthesis terminates when the ribosome
translocates to a termination codon.
• There are no tRNAs with anti-codons
complementary to the termination codons.
• No tRNA enters the A site of the ribosome when
a termination codon is encountered.
• Instead, proteins called release factors bind to
the ribosome (E. coli has 3 release factors—
RF1, RF2, & RF3).
o RF1 recognizes the termination codons UAA & UAG,
o RF2 recognizes UGA and UAA.
o RF3 forms a complex with GTP, binds to the ribosomes
and stimulates RF1 & RF2.
• The release factors then promote the cleavage of
the polypeptide chain & the tRNA from the P site.
• Additional factors then release the tRNA from
the P site & the mRNA from the ribosome
 Stages of eukaryotic translation
• The general synthesizing system is similar to that in prokaryotes with the
following differences:
1. The sequence of binding of the raw materials is different and more than three
initiation factors have been identified
2. The first amino acid Methionine is not formylated
3. Smaller 40S subunit binds to mRNA only after binding with tRNAmet
Initiation
 GTP-eIF2 + tRNAmet GTP-eIF2- tRNAmet
 GTP-eIF2- tRNAmet+ 40S GTP-eIF2- tRNAmet-40S (43S initiation complex)
 43S initiation complex binds to mRNA at 5’ end with the help of the cap. The
reaction requires the presence of eIF3 assisted by eIF4A,4B and 4F and ATP
GTP-eIF2-tRNAmet-40S + mRNA GTP-eIF2-eIF3- tRNAmet-40S-mRNA
• The complex then moves to locate the AUG and associates with the 60S to from
the 80S complex.
• GTP-eIF2-eIF3-tRNAmet-40S-mRNA + 60S tRNAmet-80S-mRNA + eIF2-GDP
+eIF 3+ P
 Stages of eukaryotic translation (contd.)
Elongation
• The process is similar to that in prokaryotes but instead of EF-Tu is substituted
by EF-1 and EF-G is substituted by EF-2.
Termination
• This process is also similar to that in prokaryotes but only one release factor is
identified called eRF1.
Post translational processing
• It is the final stage of protein synthesis that takes place after translation.
• The protein first attains its native conformation by forming H bonds, Van der
Waal’s linkages, hydrophobic interactions, etc. Then, it is processed into its
biologically active form. Some of the post-translational modifications are:
1. Amino terminal modifications: fmet (in prokaryotes) & met (in eukaryotes)
2. Glycosylation: attachment of carbohydrate side chains to form glycoproteins
3. Addition of prosthetic groups: proteins require for their activity covalently
bound prosthetic groups. Example the heme group of hemoglobin
Post translational processing (contd.)
3. Proteolytic processing
• large, inactive precursor polypeptides that are proteolytically trimmed to form
their smaller, active forms. Examples proteases such as chymotrypsinogen
& trypsinogen.
4. Formation of disulfide bridges
• After folding, some proteins form disulfide bridges between Cystine residues.
• disulfide bonds are common in proteins to be exported from cells
• this way help to protect the native conformation of the protein molecule from
denaturation in the extracellular environment
The genetic code