The Nucleolus, Ribosomes and Protein Synthesis

Professor Alfred Cuschieri

Department of Anatomy, University of Malta





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. 4th Edition

Chapter 19 Gene Expression: I. The Genetic Code and Transcription

Chapter 20 Gene Expression: II. Protein Synthesis and Sorting






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)







  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 5’to 3’ direction




Is the process whereby the genetic message for a specific protein is transcribed on to m-RNA 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



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 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.  





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 codon CAU on m-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 GTP

















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.




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 r-RNA 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



 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



 Inhibition of peptidyl transferase site

e.g. Chloramphenicol ;   Lincomycin


 Prevents translocation of the protein from A to P site   






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