The Nucleolus, Ribosomes and Protein Synthesis

Professor Alfred Cuschieri

Department of Anatomy, University of Malta
Objectives
State the main characteristics of messenger, ribosomal and transfer RNA
Distinguish between transcription and translation
Describe the composition and structure of the nucleolus
Explain what constitutes the nucleolus organizer regions
Outline the role of ribosomes in protein synthesis
Discuss how antibiotics inhibit the growth of bacteria and have different sites of action

Recommended reading
The World of the Cell: Becker WM, Kleinsmith LJ, Hardin J. 4 Edition
Chapter 19 Gene Expression: I. The Genetic Code and Transcription
Chapter 20 Gene Expression: II. Protein Synthesis and Sorting
th

The nucleolus and ribosomes form part of the protein synthesizing

machinery of the cell
The nucleolus is the site where most of the ribosomal RNA (r-RNA) is
transcribed
Ribosomes are composed of r-RNA and proteins
The ribosomes are the sites where protein synthesis occurs
Synthesis of specific proteins (transcription) also requires the action of two other
types of RNA:
m-RNA as a template
t-RNA for the assembly process

The Cell Has 3 Types of RNA

messenger RNA (m-RNA)
-there are about 105 varieties; each corresponds to a gene; each carries a coded
message to cytoplasm
ribosomal r-RNA (r-RNA)
there are 4 varieties; 4 RNA constituents of ribosomes
transfer t-RNA (t-RNA)

 there are 20 varieties corresponding to 20 amino-acids; they transfers amino

acids to polypeptides chain

m-RNA is the transcript of a gene

DNA has two strands. The template strand carries the message and is transcribed to
m-RNA.
5' --- T G T A C G A T T C C G A T G A C T --------3'

coding strand

3' --- A C A T G C T A A G G C T A C T G A --------5'

template strand

5' --- U G U A C G A U U C C G A U G A C U
--------5'

m-RNA

codon codon codon codon codon codon

A1 --- A2 --- A3--- A4 --- A5 --- A6-----

amino acid chain (Polypeptide)

Note:
M-RNA is translated to protein. A triplet of bases on m-RNA is a codon and corresponds
to an amino acid.
m-RNA is similar to the coding strand except that T is replaced by U.
transcription occurs in the 5' to 3' direction; nucleotides are always added at the 3 end
translation also proceeds in the 5to 3 direction

Transcription
Is the process whereby the genetic message for a specific protein is transcribed on to mRNA using the transcribed strand of DNA as template.
Occurs in the nucleus of eukaryotic cells
Occurs in the 5 to 3 direction
The sequence of bases on m-RNA:
Is complementary to that on the transcribed strand of DNA
Corresponds to that on the coding strand except that U replaces T

Translation
Is the process of synthesis of a specific protein using m-RNA as a template.
Occurs in the ribosomes in the cytoplasm
The message is read in codons (triplets of nucleotides) in the 5 to 3 direction
Each codon corresponds to an amino acid according to the genetic code

THE GENETIC CODE

The genetic code is, by convention, interpreted with reference to the sequence of
bases on m-RNA.
The sequence of bases on m-RNA corresponds to that on the coding strand of
DNA, except that U in RNA replaces T on DNA.
The sequence of bases on m-RNA determines the exact sequence of amino acids
in the protein.

The Genetic Code

Note that there are:
4 bases - A, U, C, G;
64 possible codons;
20 amino acids
The genetic code is
degenerate i.e. One amino
acid may be represented
by more than one codon
The codon AUG codes
for methionine but it may
also serve as a"start" signal
indicating the beginning of
the coded message.
The
codons UAA,
UAG, UGA do not code
for any amino acid but act as "stop" signals for the end of a gene message.

t-RNA
Consists of a single strand of RNA folded in the form of
a cross
Has an anti-codon at one end (triplet complementary to
sequence of a codon)
Has the corresponding amino acid at the opposite end
In this example the anticodon GUA corresponds to the
amino acid histidine and the codonCAU on m-RNA

r-RNA
Is involved in the bio-synthesis of ribosomes together with proteins
4 types of r-RNA: 5S, 5.8S, 18S, 28S (distinguished by the sedimentation coefficient)
is transcribed from multiple copies of DNA
(unlike m-RNA transcribed from a unique gene)

is synthesised in the nucleolus

The Nucleolus
Consists of two parts:
-fibrillar part - consists of chromatin:(DNA transcribing r-RNA)
-granular part consists of ribonucleoprotein particles (r-RNA + proteins)

The nucleolar DNA

The genes for 5.8S, 18S and 28S r-RNA form a gene cluster
they are all transcribed together forming a r-RNA complex of 45S
these genes are present on the nucleolus organiser regions on the satellites of
the acrocentric chromosomes 13, 14, 15, 21 and 22

Note: The gene for 5S r-RNA is located on chromosome 1 and is transcribed

separately

The nucleolus organiser regions contain many repeated copies of the r-RNA gene
cluster
This
is an example of gene amplification
Multiple copies of r-RNA are transcribed simultaneously
This forms a feather arrangement: the stem is the transcribed strand of DNA;
the side strands are the forming RNA of various lengths depending on how much
of the strand has been transcribed.

Procedure for Isolation of Ribosomes

1. Prepare cell homogenate
2. Differential centrifugation to separate RER layer
3. Treat with detergent to remove membranes
4. Differential centrifugation to separate ribosomes from debris
5. Treat with low [Mg2+] - cleaves ribosomes into small and large sub-units

Composition of Ribosomes
80 S ribosomes can be broken down into two sub-units by adjusting
Mg 2+ concentration:
Small sub-unit - 40 S
Large sub-unit - 60 S

Structure of a Ribosome

Groove for binding of m-RNA

Amino-acyl site (A) for binding to next t-RNA
Peptidyl transferase site (PT)
for binding of amino acids by peptide bonds
Peptidyl site (P) for the growing peptide chain

Protein synthesis involves a number of steps:

1. Initiation
a. Dissociation of ribosome subunit requiring an
initiation factor and energy derived from G
b. Formation of an initiation complex consisting of the
small subunit and the first t-RNA.
The initiator codon is AUG
and the initial amino acid is methionine

c. The ribosome is closed

- the initiation complex has a one amino acid (met)
attached to it

2. Translocation
m-RNA moves by one codon; a new t- RNA occupies the A site;
the two amino acids fuse at the PT site

3. Chain elongation
m-RNA moves by one codon; t- RNA is displaced from the P site; another enters the A site
Elongation of the polypeptide chain requires an elongation factor and GTP

4. Chain termination
A releasing factor is required for ending protein synthesis. It attaches to the terminator codon
UAG.

Polyribosomes
Free ribosomes usually occur in small clusters termed
polyribosomes.
UsuallyOneOne m-RNA runs successively through the
several ribosomes in a cluster producing multiple copies of
a protein

Ribosomes may attach to the RER

Attacment of ribosomes to RER requires:
1. A signal peptide the first part of the protein being
transcribed
2. A signal recognition particle (SRP) attaches to the
signal protein
3. A docking protein to bind to the SRP
4. Ribophorin - a membrane protein forms a channel for the
signal peptide to enter the lumen of the RER

Many Antibiotics act by Inhibiting Protein Synthesis in Bacteria.

Bacterial (prokaryotic) ribosomes are similar in their action to mammalian ribosomes but differ
their sedimentation coefficient (70S consisting of 30S and 50S subunits), the details of the rRNA and of the associated proteins .

Antibiotics act at various sites in the protein synthesis pathway. The following are
some examples:
Inhibiting the attachment of the first t-RNA to the small subunit
e.g.Streptomycin
Iinhibiting the binding of t-RNA to A-site
e.g.Tetracycline; Fusidic acid
Blocking protein synthesis by bind to the A site (molecular analogue of t-RNA)
e.g. Puromycin
Preventing ribosome assembly by binding to the large (50S) subunit
e.g.
Eryhthromycin
Inhibition of peptidyl transferase site
e.g. Chloramphenicol ; Lincomycin
Prevents translocation of the protein from A to P site
e.g.Thioseptrin

Why do antibiotics inhibit protein synthesis in bacteria but not in humans?

Many antibiotics are specific for prokaryotic (bacterial) ribosomes (70S)
Prokaryotic ribosomes (70S) have a different r-RNA and protein composition from
eukaryotic (80S) ribosomes
The bacterial cell wall is permeable to some antibiotics while the plasma membrane of
eukaryotic cells is not

Some inhibitors of protein synthesis also act on eukaryotic cells

Substances that inhibit protein synthesis in human cells are lethal to cells that are
actively dividing
Such substances are useful as cytostatic agents e.g. cycloheximide

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