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.
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
- Secondary structures of each of the rRNAs have been determined by their sequence analysis.
- The 16s rRNA and 23s rRNA, each of them, show four domains and each of them are distinguished by the binding of specific riboproteins.
- The 16s RNA’s domain I starts from 5’ end progresses into domain II, III and IV in an order.
- At the 3’ end of the IV th domain of the 16s rRNA it has a small segment with a sequence that binds to the 5’ end of non coding Shine-Delgarno sequence which is a leader sequence of mRNAs.
- The sequence is 3’ AUUCCUCCACUAG—5’.
- Similarly there are specific ribo-protein binding sites in each of the domains, ex. S4 & s20 bind to domain I, s8 7 & s15 bind to domain II, s7, 9,13 and 19 bind to domain III; thus each of the binding domain can be identified.
- Binding of tRNA and other factors to specific regions have been discerned by a variety of techniques such as electron microscopy, immuno labeling, neutron scattering techniques.
- Even eukaryotic subunit rRNAs show such domains identified by their ability to bind to certain riboproteins and other RNA species such as tRNA and other translational factors.
- Some commonality of the sequence and secondary structures can be observed when one compares the 5’end of the 23s RNA of prokaryotes with that of 5’ ends of eukaryotic 5.8sRNA. A secondary structure of rRNAs suggests the overall structural features of ribosomes.
- It can be
discerned that rRNAs from eukaryotes also show similar structural
features.
Assembly:
- Assembly or association of riboproteins with rRNA is sequential and stepwise.
- Methylation of 2’OH of ribose sugars and at adenine and guanine nucleotides at specific position is critical, and such methylations are performed by specific methylases and they use sequence driven secondary structures as motifs for identification of sites.
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]
- As the 5’ end of the precursor rRNA emerges during its synthesis, s4, 8 and 15 bind to this region tightly. Then s17 and s7 join directly on to the RNA, later other proteins join by protein-protein or protein-RNA interactions.
- Proteins s1, 3, 4,5,9,12,18 and the 3’ end of 16s RNA are involved in mRNA binding.
- Peptidyl transferase function at P-site involve proteins L-2, 11, 15,16,18,23, and 27 in association with 23s RNA.
- About 40 ntds long region of 16S RNA is located in the platform of 30s ribosomal subunit.
- Peptidyl transferase occupies valley in the ribosome.
- Two L7 and two L12 together act as GTPase.
Role of rRNA in protein
synthesis (Prokaryotic):
- In the molecular organization of ribosomes, both RNA and proteins are ordered and occupy certain specific invariant positions and perform specific function. As a 3-D structure, it goes through several conformation changes with each binding events and catalysis.
- The 3’ terminus 16s RNA of 30s ribosome directly interacts with 5’ end Shine-Delgarno sequence of mRNA and facilitates initial binding of it ribosomal surface so as to bring the first codon exactly to P site.
- Specific regions of 16s RNA interact with tRNA for the binding at P and A sites.
- 23 S RNA interacts with CCA terminus of peptidyl tRNA.
- It is envisioned that there is RNA-RNA interaction and protein –protein interaction as well as protein-RNA interaction, thus both subunits are held together while they perform functions.
- There must be some proteins, which perhaps act as motor proteins in moving ribosomes on single stranded mRNA in ATP dependent manner, similar to helicase.
- Cleavage of 3’ region of 16S RNA by E3 Colicin abolishes initiation of translation.
- Methylation of Adenine at 6th position and at 3’ end (di methylation to Adenine) of 16s RNA, facilitates dimrization of 30s and 50s units. If methylases are absent dimrization fails.
- Sensitivity to Kusugamycin depends upon methylation or absence of methylation at the above-mentioned sites. Mutation at these sites abolishes sensitivity to the drug, but methylation at these sites makes it Kusugamycin resistant.
- Kusugamycin blocks initiation of translation by releasing F-met tRNA from ribosomal surface.
- Mutation in the of 3’ end of 16s RNA suppress terminator codon function.
- A mutation in rRNA can lead to frame shift function, because recognition between mRNA and rRNA doesn’t take place.
- A region at 1400th ntds in 30s, is directly involved in the binding of peptidyl tRNA at P-site.
- Both 16s and 23s RNAs are involved in organizing A and P site.
- The CCA-end of tRNA at P- site protects 23SRNA from Rnase digestion.
- The 23s rRNA is involved in organizing the exit site E, found in 50s subunit.
- The 23s RNA
is involved in catalyzing peptide bond formation; hitherto it is believed
that an enzyme called di peptidyl transferase found in larger ribosomal
subunit is involved in peptide bond formation.
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.