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

• 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.

Riboproteins (Prokaryotic):

• Riboproteins, isolated from small and large subunits, have been numbered as small and large riboproteins.
• They are numbered as s1 to s21 and L1 to L31 respectively. The numbering is based on the mobility of each of the riboprotein subunits on a 2-D polyacrylamide gel. The S1 is the largest protein found at the left top most corner of the gel and s21 is the smallest, found at the right bottom most corner of the gel.
• Each of the riboproteins from both 70s and 80s ribosomes have been purified and antibodies have been obtained and many proteins have been subjected to X-ray diffraction, immuno-diagramming, neutron scattering, electron microscopy, NMR and cross linking to other riboproteins
• In prokaryotes s20 and L26 are common to both ribosomal subunits and located at the interface of the subunits.
• L7 and L12 are found 4 copies each. Their N- terminal is acetylated and forms stable complex with L10.
• But most of the riboproteins are made in single copies.
• Amino acid sequence of most of the riboproteins has been determined, where the number of amino acids ranges from 46 (of smallest subunit at right bottom most in the gel) to 557 in the largest subunit s1 (Left top most in the gel).
• L1 is the largest with 25 KD and L34 with 45KD.
• There is sequence homology among the protein subunits, but in general, they are rich in Lysine and Arginine, both are basic amino acids.
• Structural motifs of riboproteins show 3 b sheets and 2 a helical strands and resemble that of Sn RNPS, rho, and eIf-4B. Such motifs are called RRMs (rRNA Recognition Motifs).
• Most of the riboprotein genes have been identified and their operons have been established.
• Each operon consists of several genes controlled by a single promoter element.
• In prokaryotes the riboprotein genes have been organized into seven clusters called rRNA operons.
• Some of these operons not only contain riboprotein genes but also contain other genes like RNAP subunits and translational factors.
• Their synthesis is regulated at translational level, which depends upon the availability and the abundance of rRNA segments.
• If the concentration of rRNA falls short, the translation of their mRNAs is inhibited by their own proteins by binding to specific regions of the mRNAs they prevent translation, which is called autogenous regulation.
• Subunits of riboproteins act as self-assembling systems with rRNA providing a template or threading for sequential assembly.
• As rRNA is synthesized (synthesized as a precursor RNA), it is methylated at specific sites, which acts as recognition points for processing RNA and also assembly of proteins.
• Assembly of riboprotein with rRNA is hierarchical, in the sense, some proteins bind first to certain RNA sequence derived secondary structures, then another set of proteins bind either by RNA- protein interaction or protein-protein interaction or by both.
• There is a gradual build up of the assembly line, which ultimately produces a highly organized and a dynamic structure, where the position of each protein is fixed with respect to each RNA fold or sequence.
• Though it exhibits a specific 3-D shape, it is not a rigid structure but it always undergoes conformational changes according to the binding of other translational components or regulatory factors. Structurally, as well as functionally, ribosomes are flexible and highly dynamic.
• In the overall organization of ribosomal structure, some regions don’t have any RNA but only proteins. The whole structure appears as porous; through some of RNA folds protrude to the surface.

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

2^nd level s15 I s20 s7

3^rd level s17 s13

4^th level s16

5^th level s12 s9 s19

6^th 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 6^th 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 1400^th 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.

Structural Features of Ribosomes (Prokaryotic):

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

• For initiating translation process, to begin with the small subunit should be separated from the large subunit; it is at later part of initiation both subunits join.

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 extended finger acts like a stalk and contains L7/L12 with GTPase activity.

The central protuberance contain 5s RNA.

• The valley is located between the blunt thumb like projection and central protuberance. The ridge or thumb like portion contains L1/9.

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.

• The 3’ end of the 16sRNA is located in the platform. It is through cleft region the mRNA is threaded and both P-site and A-site are located on the surface of platform and the base of the head.

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

• Peptidyl tRNA and aminoacyl tRNA binding locations (P and A sites) are at the cleft region 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.

• The stalk (finger like) of 50s ribosome contain GTPase activity.

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

• Large subunit also has sites for the binding of peptidyl (P) and aminoacyl tRNAs (A) in line with small subunit, plus it has another site for the exit (E) of decoded tRNA.

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

• ER Membrane binding domain in the large subunit is by the side of tunnel exit.

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.