Ribosomes:

Introduction:

They are also called structural RNAs for they act as structural components of Ribosome organelle.  The ribosome in its entirety is constructed on ribosomal RNA as a scaffold on which riboproteins are sequentially built to produce a highly dynamic structure, which has astounding abilities to function as translation machine. 

 

An excellent over view of ribosomal subunits hugging to each other.

 

Distribution:

 

Ribosomes are found in almost all organisms except viruses.  An E.coli cell may contain 15000 to 20000 ribosomes at any given time, but an active eukaryotic cell may have 10-20 times the number of prokaryotic cells.  Oocytes of certain amphibians’ posses’ three million ribosomes per cell and the same is stored for the future use.  While in prokaryotes, ribosomes are distributed through out the cell, eukaryotic cells contain different classes of ribosomes and they are located in different sites like cytoplasm, mitochondria and plastids.  Cytoplasmic 80s ribosomes are either bound to endoplasmic membrane or freely.  The majority of the so-called free ribosomes are found located in the intersection of microtrabacular(?) and actin filament network.  On the contrary cellular organelles like chloroplast and mitochondria contain another class of ribosomes called 70s, which are more or less similar to that of bacterial ribosomes.  In the Oocytes of chicks and lizards, ribosomes are aggregated on membranes into crystalline structures.  They remain inactive till they are required at some stage of development.

Class of ribosomes:

Ribosomes can be isolated by magnesium precipitation. If some ribosomes, obtained from a eukaryotic organism, are subjected to density gradient ultracentrifugation, ribosomes settle into two distinct bands.  Based on the sedimentation values, determined by Svedberg, they can be distinguished into 70s and 80s ribosomes.  The 80s ribosomes are found in cytoplasm, whereas 70s types are found in mitochondria and chloroplasts.  The 70s type are smaller and 80s are little larger.  However, prokaryotes contain only one kind of ribosomes i.e. 70 type.  The 80s and 70s ribosomes can be further distinguished by their sensitivity to chloramphenicol (CAP) and cycloheximide (CHI). The 70s ribosomal mediated protein synthesis is inhibited by chloramphenicol, while 80s ribosomal protein synthesis is inhibited by CHI.

 

Chemical composition:

Components of Ribosomes:

 

Types

RNA size

Number of proteins

Methylations

Functions

70 S ribosomes

 Coded by seven genes

 

 

30 or more methylations

30s subunits

16s RNA,

1540-42 ntds

21 (s1 to s21)

10 at 2’OH,

2,methyl adenines,

2,dimethyl guanines

Help in processing and folding

50S subunits

23s RNA,

2900 ntds;

5s RNA,

120 ntds

31, L1 to L31

20 at 2’OH of sugars

 

80S ribosomes:

Coded by hundreds of genes located on chromosomes12,13,14,21 and 22

 

 

>100 sites for methylations and 100 sites for pseudouridenylations

Yeast has 43 pseudo uridines

40S subunits

18s RNA;( 1843

Or 1900 ntds)

33;

S1 to s34

43 to 44 methylations at 2’OH groups, plus conversion of Uridine into pseudo-Uridines

 

60s subunits

28s-RNA;(4718- 4800 ntds);

5.8s RNA;(160ntds);

5s RNA;(120ntds);

 

49;

L1 to L45-50

74 methylations at 2’OH of sugars,

Methylation at adenine,

Methylation at guanine, plus conversion of Uridine into pseudo-Uridines

 

Mitochondrial ribosomes: 70s like (general);

Fungus-73s;

Maize-78s;

28s

12s

-1560 ntds,48 proteins

-29 proteins

 

 

 

Chloroplast ribosomes: 70s

16s RNA

23sRNA,

5s RNA,

4.5s RNA

 

 

 

 

 

 

 

 

Prokaryotic Ribosomal RNA and Riboproteins:

 

 

This figure shows 70S ribosomal subunits

 

 

 

This is simple diagram showing the possible secondary structure based on nucleotide sequences

 

 

              A simple diagram showing subunit components

 

 

 

 

Riboproteins (Prokaryotic):

 

 

Assembly:

 

 

 

 

The ribbon diagram shows the positioning of tRNA on large ribosomal surface; A,P and E sites

 

 

 

Assembly of small ribosome subunits:

16sRNA + 16 s riboproteins à 21 s particles (can assemble at 20^oC),

21s particles + 6s riboproteins >à 26 s particles,

26 s particles ----> 30 s particles.

 

Assembly of Large Ribosome subunits:

 

23SRNA + 5sRNA -à 33 s particle,

33 s [articles -à 41 s particles,

41 s particles -à 50s particles

 

 

During dissociation also, certain subunits dissociates fast, even at the earliest steps of preparation; they are called split proteins. Such proteins are found both in small and large subunits.  Even during assembly, certain proteins associate at 0^oC, this is because great affinity of some proteins to certain RNA sequence.  Cold sensitive mutants block such assembly; they are called Subunit Assembly Defective mutants (SAD mutants).  Proteins, which associate, first are hard to disassociate and they are called core groups, and proteins, which assemble last, are the first to dissociate.  The following figure depicts sequential steps in the assembly.

 

 

rRNA          5’--------------------------------------------------------------------------3’

                       I          I           I        I

1st level                    I          s4      I    I     s8

2nd level                   s15     I          s20          s7

3rd level                         s17             s13

4th level      s16

5th level                         s12            s9  s19

6th level                   s18                              s5

 

 

 

Assembly sequence:

                    

30s =  17.5sRNAàs4,s8,s15-às1,s5,s7,s13--->s2,s3,s6,s9,s10

s17, s20        s16, s21   s11, s12, s14, s18/19

 

50s= 25sRNA--->L1,4,5,8,9,10---->L3,7,11,14-->L2, 6,12,10,28,31,32,

                     13,17,18,20, 15, 19, 23      

                     21,21,22,23,

                     24,25,27,29,

                     30, 33.

 

   30s [16s RNA]        O^oC        40^oC                  O^oC

+[ s21  proteins]--------------------> 21s--------------->26s------------->30s

                                 

 

 

50s [23sRNA]     o^oC               44^oC           O^oC          50^oC

+5sRNA+34L] ---------------->33s---------------->41s------------->48s----------->50s

Proteins]   

 

 

Role of rRNA in protein synthesis (Prokaryotic):

 

 

Structural Features of Ribosomes (Prokaryotic):

 

Structurally prokaryotic ribosome has 200 x 220 A^o dimension and the size of eukaryotic ribosome is slightly larger. 

The larger subunit looks like a cup shaped palm having a central protuberance curved inwards, a blunt thumb like structure and a last finger like structure projecting outwards. 

 

The central protuberance contain 5s RNA. 

Actually the valley provides peptidyl transferase activity. 

The large subunit has a narrow tunnel like region, which extends from peptidyl assembly site to exterior, through which nascent polypeptide chain is threaded through with NH3+ end ahead.

 

 

 

 

 

 

This is ribbon model of the large ribosomal subunit showing proteins in yellow color and RNA in grey shades, the lower is another version of the same.

 

 

The length of the tunnel can hold about 25 to 30 amino acid long polypeptide chain and has the diameter to accommodate the chain. 

It is at the posterior end, where polypeptide chain exits, contains a site for the binding of large ribosome to endoplasmic reticular membrane.

 

This diagram shows a tunnel through which the nascent polypeptide threads through as it is translated.

 

 

The small subunit is split in the top region into a platform and a head; the space between them is called cleft. 

Ribosomal site for the binding of mRNA to 16sRNA and the binding of initiation factors are located in the platform of 30s ribosome. 

The small ribosomal subunit has an additional site called A site to the right of A site, where the incoming aa tRNAs are screened.

Peptidyl transferase activity is located in the valley of large unit.

The tunnel is 100-120 A^o long, 25A^o broad and can hold approximately 20-30 amino acid long polypeptide chain.

 

Ribosome Mediated Inhibitors of translation:

 

Kusugamycin: initiation (PK), displace F-met tRNA, mutants lack methylation of 16 s rRNA at the 3’end.

Streptomycin: initiation (PK), mutation in s12 of 30s ribosome causes resistance.

Kirromycin:  elongation (PK), EF-Tu-GDP release is blocked by the antibiotic and no recycling.

Puromycin:   elongation (PK), premature termination, because Puromycin has structure similar to tRNA configuration.

Erythromycin: peptidyl transfer (PK), blocks peptide bond formation, mutation in 23sRNA results in resistance.

Chloramphenicol: peptidyl transfer (PK), blocks peptidyl bond formation,

Cycloheximide: translocation (EK), inhibits peptidyl transferase on 60s subunit.

Fusidic acid:       translocation (PK),  EF-G-GDP cannot be released, no recycle.

Thiostrepton: translocation (PK) binds to 23sRNA and inhibits GTPase activity.