Ribosomes:

Introduction:

Ribosomes are made up of rRNAs and proteins 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.www.library-online.blogspot.com; http://urei.bio.uci.edu/;https://sites.google.com

 

http://www.ck12.org/

Three-dimensional views of the ribosome, showing rRNA in dark blue (small subunit) and dark red (large subunit). Lighter colors represent ribosomal proteins.;www.scienceprofonline.com, en.wikipedia.org; www.biology.about.com

 

Diagram showing the translation of mRNA and the synthesis of proteins by a ribosome;www.princeton.edu; chemistry.about.com

www.scc.acad.bg

 

Ribosome binds to both mRNA and tRNA; structural features of tRNAs at different positions is shown; Binding of tRNA during translation,  binding positions such as E, P and A sites are shown. www.oregonstate.edu;www.chegg.com

 

http://oregonstate.edu/

 

www.faculty.samford.edu

 

 

 

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, perhaps in millions.  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 loci like cytoplasm, mitochondria and plastids.  Cytoplasmic 80s ribosomes are either bound to endoplasmic membrane or free.  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 themselves contain another class called 70s ribosomes, 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) respectively. 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 several 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, rRNA genes located on chromosomes 12,13,14,21 and 22 (in humans)

 

 

>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;

16s

12s

-1560 ntds, 48 proteins

-29 proteins

 

 

 

Chloroplast ribosomes: 70s (50s and 30s)

23s RNA

16s RNA

4.5s RNA?

23sRNA,

5s RNA,

4.5s, 16s RNA

 

 

 

 

 

 

 

 

Prokaryotic Ribosomal RNA and Riboproteins:

 

 

                             This figure shows 70S ribosomal subunits

 

Prokaryotic and Eukaryotic Ribosomes

                             A simple diagram showing subunit components of ribosomal subunits www.is.muni.cz

 

 

 

                                           Fig 1 full size

In vitro assembly of 30s ribosomes showing rRNA; Primary and secondary ribosomal proteins in black and tertiary proteins in white.   S6 and S18 heterodimers are enclosed in box; b. secondary structures of 16S rRNA one can observe four domains 5’ central , 3’ major and 3’ minor domains are in green, red and gray; C. schemating representation of in vitro 30S ribosomal assembly. Kristi L Holmes & Gloria M Culver; http://www.nature.com/

                                                                             

 E.coli 23S rRNA secondary structural features; http://rna.ucsc.edu/

 

 

 

 

 

Full-size image (28 K)Full-size image (28 K)

Secondary Structure Diagrams of the 23S and 16S rRNAs: The different domains are color coded. (a) The 23S rRNA of H. marismortui. (b) The 16S rRNA of T. thermophilus. Locations of helix 44 and the peptidyl transferase center (PTC) are indicated. Diagrams were modified with permission of R. Gutell (http://www.rna.icmb.utexas.edu)

 

 

 

Riboproteins (Prokaryotic):

 

 

Assembly:

 

 

 

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

 

Next, let’s put red circles around the ribosomal proteins for which there is experimental evidence to support a moonlighting role (the moonlighting roles are listed below the

 

ribmooncons

http://designmatrix.wordpress.com/

 

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]          

 

 

i-bec1593ca6d82958866bbda92e04d296-SRP1.jpg

 

The large subunit of the ribosome is in blue, the small subunit in yellow. The canal in the large subunit is where the newly synthesized peptide (protein) is pushed through (think of it as the birth canal of the ribosome. As new amino acids are being added to the top where the green blob is (the green blob is a tRNA – I will not explain it here any further) the newly synthesized peptide (in green) is being pushed out, or down in this diagram, towards the exit. At the exit site, SRP (here in red) sits and in this diagram is holding the part of the polypeptide that encodes the signal sequence (the green cylinder). SRP is also making contacts to two subunits of the ribosome (the two orange blobs). When bound to the signal sequence SRP makes a total of four contacts to the ribosome. Some of these contacts are shared with another ribosome cofactor, trigger factor (TF), which acts as a chaperone for the newly emerging nascent chain. Since TF and SRP share binding sites they may be mutually exclusive. In fact when SRP holds on to a newly made signal sequence and engages the SRP receptor in the ER, TF is known to be released from the ribosome. http://scienceblogs.com/

 

www.micro.magnet.fsu.edu

 

Image result for tRNAs bound to large ribosomal surface

The ribbon diagram shows the positioning of tRNA on large ribosomal surface; A, P and E sites; http://liberary-online.blogspot.com

 

 

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.

 

 

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.

http://dwb.unl.edu/Teacher

This diagram shows a tunnel through which the nascent polypeptide threads through as it is translated. http://www.bioss.uni-freiburg.de/

 

 

 

 

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.

 

Eukaryotic Ribosomes:

 

                                                         

http://10e.devbio.com/

 

http://www.nobelprize.org/

Promoter regions of Human MRP, P and U6.A. Organization of pre-rRNA based on [37]. In bacteria rRNA genes are co-transcribed as a polycistronic precursor (although exceptions are common). Most eukaryotes vary only in the length of their ITS regions, an extreme case being the microsporidian Encephalitozoon cuniculi which has completely lost its ITS2 having a fused 5.8S/28S subunit. RNase P and RNase MRP do not cleave the main transcripts but trim the ends of their respective substrates (the tRNA or 5.8S rRNA) after cleavage by other enzymes. In eukaryotes the 5S rRNA is transcribed separately by RNA polymerase III. B. The Diplomonad Giardia lamblia has the usual order of rRNA subunits with short ITS regions, however RNase MRP has not yet been characterized from this species. RNAstructure folding of G. lamblia ITS1 [56] showing a single stranded region between two stem loops that could possibly be an A3 site. Other foldings of this sequence and foldings of other sequences (DQ157272 and AF239841) produce just a single stem-loop.;http://www.biomedcentral.com/

 

Prokaryotes contain ~ 70,000 ribosomes per cell.  Eukaryotes contain more ribosomes approximately 8-10 million per cell, but human oocyte contains hundred times more than the normal number found in cells. Ribosomes in eukaryotes are larger 80S than prokaryotes 70S. Their genes are found in multiple copies and located only in certain chromosomes, far example in Eukaryotes their rRNA genes are found in secondary constriction region of chromosomes 13,14, 15 21 and 22.   At the beginning of cell cycle chromosomal complex opens up and rDNA unwinds to allow transcription of rRNA gene which are organized in tandem repeats with intervals non transcribed space.  The primary transcript is large and during initial folding snoRNA dependent cleavage of primary transcript takes place. Few other sno RNAs are involved modification of specific nucleotides in rRNA.  This RNA now get associated with riboproteins in step by step manner, specific riboproteins by proteins assemble, while assembling 60s ribosome also get associated with another rRNA called 5S RNA.  Process of transcription and assembly of proteins with processed rRNA takes place in a specific region called nucleolar organizer.

Assembly of ribosomal proteins with RNA and protein takes place in hierarchical mode.

 

http://nar.oxfordjournals.org/

 

rRNA gene –precursor rRNA processing into smaller fraction; www.quzzlet,com; http://biosiva.50webs.org/

 

http://www.mcqbiology.com/

www.tumbler.com

Composition:

Compared to their prokaryotic homologs, many of the eukaryotic ribosomal proteins are enlarged by insertions or extensions to the conserved core. Furthermore, several additional proteins are found in the small and large subunits of eukaryotic ribosomes, which do not have prokaryotic homologs. The 40S subunit contains a 18S ribosomal RNA (abbreviated 18S rRNA), which is homologous to the prokaryotic 16S rRNA. The 60S subunit contains a 26S rRNA that is homologous to the prokaryotic 23S ribosomal RNA. In addition, it contains a 5.8S rRNA that corresponds to the 5' end of the 23S rRNA, and a short 5S rRNA. Both 18S and 26S have multiple insertions to the core rRNA fold of their prokaryotic counterparts, which are called expansion segments. For a detailed list of proteins, including archaeal and bacterial homologs please refer to the separate articles on the 40Sand 60S subunits.

 

Eukaryotic[3]

Bacterial[3]

Ribosome

Sedimentation coefficient

80 S

70 S

Molecular mass

~3.2*106 Da

~2.0*106 Da

Diameter

~250-300 Å

~200 Å

Large subunit

Sedimentation coefficient

60 S

50 S

Molecular mass

~2.0*106 Da

~1.3*106 Da

Proteins

47

33

rRNAs

·                28 S rRNA (3354nucleotides)

·                5 S rRNA (154 nucleotides)

·                5.8 S rRNA (120 nucleotides)

·                23S rRNA (2839 nucleotides)

·                5S rRNA (122 nucleotides)

Small subunit

Sedimentation coefficient

40 S

30 S

Molecular mass

~1.2*106 Da

~0.7*106 Da

Proteins

32

20

rRNAs

·                18S rRNA (1753 nucleotides)

·                16S rRNA (1504 nucleotides)

                                                          https://en.wikipedia.org

www.biologyexams4u .com

 

Specific Ribosomal proteins and specific functions:

 

It is difficult to assign specific function to specific ribosomal proteins for all of them have cooperative action, yet some have specific functions. Ribosomes contain rRNA 28s, 5.8s and 18s rRNAs of about 120-4500 ntd long with 50-80 riboproteins of 25-300 a.a. in eukaryotes.  Accuracy of transcriptions is  in order of 1:20 000 to 1: 60 000 and translational accuracy is about is about 1:3000.  E.coli ribosomes contain 5S rRNA (120ntds), 23s -2907 ntds and 16s-1542 ntds.  Large ribosomes contain 36 proteins (L1-L36) and small subunit contains 21 subunits of proteins (S1-S21), these numbers derived form protein analysis on PAGE.  All protein are single copy proteins with the exception of L7/L12.  L7 is acetylated  form of L12 and together with L10 organize into pentameric complex.  Most of these protein’s tertiary structure has been established.  Even the position of protein with respect to 3-D organizes rRNA.  Sequential association is shown above in the page 12 and 13.  Identified riboproteins , some are characteristic  for all domains of life and some are characteristic proteins in bacteria, Archaea and eukaryotes. Ribosomal signatures, idiosyncrasies in the ribosomal RNA (rRNA) and/or proteins, are characteristic of the individual domains of life. One finds correlations between the rRNA signatures and signatures in the ribosomal proteins showing that the rRNA signatures coevolved with both domain-specific and universal ribosomal proteins (Ulrich Stelzl, M eta l;Korobeinikova. AV etal; Roberts etal).

 

S5 may also have a role in translocation

L2 and Peptide Bond Formation

Proteins of the Ribosomal Stalk: Factor Binding,

L11: binding of deacylated tRNA to the A site and the ‘GTPase associated centre’,

L9: Involved in the tRNA Arrangement at the P Site?,

Ulrich Stelzl; http://www.javeriana.edu.co/

 

 

Ribosome Mediated Inhibitors of translation:

 

Binding sites of inhibitors on the yeast ribosome.

Binding sites of inhibitors on the yeast ribosome: The binding sites can be grouped in four functional regions: the tRNA E-site and the peptidyl transferase centre (PTC) on the large subunit (60S) and the decoding centre (DC) and the mRNA channel on the small subunit (40S).  Twelve eukaryotic specific and 4 broadband spectrum inhibitors; all inhibitors were found associated with messenger RNA and transfer RNA binding sites. In combination with kinetic experiments, the structures suggest a model for the action of cycloheximide and lactimidomycin, which explains why lactimidomycin, the larger compound, specifically targets the first elongation cycle. The study defines common principles of targeting and resistance, provides insights into translation inhibitor mode of action and reveals the structural determinants responsible for species selectivity which could guide future drug development. Irina Prokhorova et al; http://www.nature.com/http://www.nature.com/

 

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.