Chapter 14 notes

Translation of mRNA depends on Ribosomes and Transfer RNAs

-Translation of mRNA is the biological polymerization of amino acids into
polypeptide chains
-The process occurs only in association with ribosomes which serve as
nonspecific workbenches
-The discovery of tRNA was correlated with the question regarding how the
triplet codons of mRNA direct specific amino acids into their correct
position in the polypeptide
-tRNA adapts genetic information present as specific triplet codons in
mRNA to their corresponding amino acids, using three
consecutive
ribonucleotides complementary to a triplet codon on the
mRNA,
called the anticodon which again can base pair with the
triplet codon
-Another region of the tRNA is bound covalently to the codons
corresponding amino acid
-Largely with the help of hydrogen bonding of tRNAs to mRNA, peptide
bonds form between amino acids that are located in proximity to each other
-Using this process over and over again as the mRNA runs through the
ribosome, amino acids are polymerized into a polypeptide
-Remember, Ribosomes translate mRNA in the 5 3 direction, reading
each triplet codon and assembling the amino acids in the order specified by
the codons
(Summary) Ribosomes in bacteria and eukaryotes perform three tasks:
1. Bind mRNA and identify the start codon, where translation begins
2. Facilitate complementary base pairing of mRNA codons and the corresponding
tRNA anticodons
3. Catalyze formation of peptide bonds between amino acids on the growing
polypeptide chain

Familiarize yourself with this picture

Important regions of Ribosomes

-The aminoacyl site (A-site) binds a new tRNA molecule containing an
amino acid to be added to the growing polypeptide chain
-The peptidyl site (P-site) holds the tRNA to which the polypeptide is
attached
-The exit site (E-site) provides an avenue for exit of the tRNA after its amino
acid has been added to the chain
-Ribosomes also form a channel from which the polypeptide chain emerges
Ribosomal structure
-Eukaryotic cells have more ribosomes than bacteria
-The subunit and rRNA components are most easily isolated and
characterized on the basis of their sedimentation behavior in sucrose
gradients

-S stands for Svedberg coefficient which is a measure of their rate of

migration when centrifuged, reflecting their mass, density, and
shape
-Sedimentation coefficients reflect differences in the rate of migration
of different sized particles and molecules, S.C. are not additive
-Not Additive? Means that for ex. The 50S and 30S subunit
combine to form the 70S
-Bacterial ribosomes
-40nm at its largest diameter
-Consists of two subunits, one large 50S subunit and one small 30S
subunit
-Each subunit consists of one or more rRNA molecules and an
array of ribosomal proteins
-Large subunit- 23S rRNA, 31 proteins, 5S
-Small subunit- 16S rRNA, 21 proteins
-Combination of the 50S and 30S subunit results in the monosome
70S (molecular weight of 2.6 x 10^6 daltons)
-Contain the P- A- and E- Sites and the polypeptide channel
-Eukaryotic ribosomes
-Consists of two subunits, one large 60S subunit and one small 40S
subunit
-Each subunit consists of one or more rRNA molecules and an
array of ribosomal proteins
-Large subunit- 28S rRNA, 46 proteins, 5S, 5.8S rRNA
-Small subunit- 18S rRNA, 33 proteins
-Combination of the 60S and 40S subunit results in the monosome
80S (molecular weight of 4.3 x 10^6 daltons)
-Contain the P- A- and E- Sites and the polypeptide channel
-Main Concept
-Ribosomes are composed of two subunits, the large ribosomal
subunit and the small ribosomal subunit
-Ribosome compositionnumber and sequence of rRNA molecules
and number and type of proteinsdiffers between bacteria and
eukaryotes

-Ribosomal subunit size is measured in Svedberg units (S), a property

based on size, shape, and hydration state
-Function of RNA components
-Perform all-important catalytic functions associated with translation
-Many ribosomal proteins thought to promote binding of various
molecules involved in translation (fine tune the process)
-Degree of redundancy
-Molecular hybridization studies have established the degree of
redundancy of the genes coding for the rRNA components
-Prokaryotes
-Ex. E. Coli
-Seven copies of a single sequence encode components of 23S,
16S, and 5S
-The initial transcript of each set of these genes produces a 30S
RNA molecule that is enzymatically cleaved into these
smaller
components
-Why is this important?
-The coupling of the genetic information encoding these
three rRNA components ensures that, following
multiple transcription events, equal
quantities of all three
will be present as ribosomes are
assembled

various

-Eukaryotes
-Many more copies of a sequence encoding the 28S, 18S, and
5.8S components are present
-The initial transcript of each are transcribed to produce
45S molecules which is processed into the three smaller
components of 28S, 18S, and 5.8S
-The rRNA genes, called rDNA, are part of the moderately
repetitive DNA fraction and are present in clusters at
chromosomal sites
-Each cluster consists of tandem repeats, with each unit
separated by a noncoding spacer DNA
-Usually in humans these clusters are localized near the
ends of chromosomes 13, 14, 15, 21, 22

-5s rRNA component of eukaryotes is not part of this

larger transcript, located on chromosome 1
tRNA Structure
-tRNA molecules are the best characterized RNA molecules
-Composed of only 75-90 nucleotides
-Display nearly identical structure in bacteria and eukaryotes
-In bacteria and eukaryotes, tRNAs are transcribed from DNA as larger
precursors which are cleaved into mature 4S tRNA molecules
-Cognate amino acid- superscript above tRNA identifying the specific tRNA
by the amino acid that binds to it (ex. tRNATry)
-Robert Holley
-Discovered that # of nucleotides are unique to tRNA, each containing
socalled
modified
bases
-Each
of the
nucleotides
below
contained a
four nitrogenous bases expected in RNA (G,

modification of one of
C, A, U)
-Inosinic acid (containing purine hypoxanthine), ribothymidylic acid,
and pseudouridine are the modified structures that were created
after
transcription of mRNA, representing posttranscriptional
modification
-Bases may be modified via enzymatic reactions to enhance hydrogen
bonding efficiency during translation
-Familiarize yourself with these bases and names
-Cloverleaf model of tRNA
-Holleys sequence analysis led him to propose the two dimensional
cloverleaf model

-tRNA has a secondary characteristic structure created by base pairing

-By arranging with paired stems and unpaired loops, the linear
sequence could be stretched in a way that would result in
several base
pairs
-The model accounted for the modified bases that generally did not
form base pairs

these
(I for

-Holley knew that GCU, GCC, and GCA specified alanine, so he

searched for an anticodon sequence complementary to one of
codons in his tRNA Ala molecule. He found it in the form of CGI
inosinic acid which can form hydrogen bonds with U, C, A)
-Therefore the anticodon loop was established

to the

-Additional findings
-At 3 ends, all tRNAs contained the sequence 3- ACC. At the
end of this molecule, the amino acid is covalently joined
terminal adenosine residue
-At 5 ends, all tRNAs contained the 5- G nucleotide at the
other end of the molecule
-The lengths of the analogous stems and loops in tRNA
molecules are very similar
-Each tRNA contained an anticodon complementary to the
known codon for the tRNAs cognate amino acid
-All anticodon loops are present at the same position of model
Three dimensional structure
-Ribosomes are 25 nm in diameter and powerful imaging techniques
are needed to explore their configurations
-Cryo-electron microscopy uses liquid nitrogen or liquid ethane to
freeze macromolecules, preserving their native state
-A frozen macromolecule is then placed on a micro-caliper and
scanned from various angles using an electron beam to create a threedimensional picture

-Geneticists speculated that

the shapes of the intervening
loops are recognized by the
enzymes responsible for
attaching amino acids to
tRNAs, called aminoacyl
tRNA synthetases (20 in total
for each amino acid)
-These enzymes are very
specific since they recognize
only one amino acid and only
tRNAs corresponding to that
amino acid (isoaccepting
tRNAs)
-Crucial detail if fidelity
of translation is to be
maintained
-Before translation can occur, the tRNA molecules must be chemically
linked to their respective amino acids which involves a process called
charging or aminoacylation

-The wobble effect allows the required min. number of different tRNA
molecules to be only 31, but there are usually more
-In the initial step of aminoacylation, the amino acid is converted to an
activated form, reacting with its carboxyl end and a 5 phosphate group of
ATP to form aminoacyladenylic acid
-This reaction occurs in association with the synthetase enzyme,
forming a complex that reacts with a specific tRNA molecule
-During the next step, the amino acid is transferred to the appropriate tRNA
molecule and bonded covalently at the 3 end adenine residue
-The charged tRNA or aminoacyl tRNA may then participate in protein
synthesis
Translation of mRNA can be divided into three steps
-Initiation
-Ribosomes when not involved in translation are dissociated
into large and small subunits
-In prokaryotes:
-Involves small ribosomal subunit, an mRNA molecule,
specific charged initiator tRNA, GTP, Mg2+, and
three
proteinaceous initiation factors (Ifs) (which
enhance the
binding affinity of various translational
components and
facilitate the process)
-The initiation codon of mRNA AUG calls for
modified amino acid N-formylmethionine (f-met)
-Steps-1. Initiation begins when the small ribosomal subunit 30S
binds near the 5 end of the mRNA and identifies the start
codon
-In prokaryotes, the binding involves a sequence of
AGGAGG that precedes or is upstream from the
initial AUG start codon of mRNA
-Sequence is called the Shine-Dalgarno
sequence and it only contains purines and is
actually 9 nucleotides long
-This sequence base pairs with the complementary region
of the 16S rRNA of the small ribosomal subunit, creating
a preinitiation complex, facilitating initiation

-A complementary pyrimidine-rich sequence is

found near the 3 end of the 16S rRNA
-Initiation factor 3 also facilitates the binding of the
mRNA to the small ribosomal subunit
-IF3 serves to inhibit the small subunit from
associating with the large subunit
2. Once the 30S subunit IF3 mRNA complex has
formed, initiation factor 2 binds to the complex,
promoting the binding of the initiator tRNA fmet to
the
complex
-Like mentioned before, the amino acid of the
initiator tRNA is fMet, so the charged
initiator
tRNA is called tRNA fMet
-IF2 plays the largest role; As a GTPase, it
interacts with mRNA and charged tRNA,
stabilizing them in the P site and
setting the
reading frame so that all
subsequent groups
of three ribonucleotides
are translated
accurately
-The initiator tRNA, carrying the first amino acid
of the polypeptide, binds to the start codon where
the P-Site will be once the ribosome is
assembled
-IF-3, IF-2, and a GTP molecule are bound to the
tRNA fMet and when IF-1 joins the complex;
together these form the 30S initiation
complex
-IF1 blocks the A site from being bound to a
tRNA
-IF1 may also be important after translation
is completed for the dissociation of the
complex
-IF3 released before binding of large subunit

subunits,

3. IF2 hydrolyzes GTP as an energy source to promote

the association of mRNA, tRNA, and the ribosomal
specifically in this case, the energy is used when

the 50S
initiation complex

In prokaryotes:

subunit joins to the 30S subunit to form 70S

-IF1 and IF2 are released
-Subsequent aminoacyl tRNA is poised to enter the A site

In Eukaryotes: