Ribosomal RNAs:

 

 

Ribosomal RNAs:

 

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.  It is deemed as the finest molecular machine.

 

An excellent over view of ribosomal subunits hugging to each other: it is like mother sitting and hugging her child. http://plantcellbiology.masters.grkraj.org/

 

Based on the system and the size of the organelles, two types of ribosomes can be distinguished i.e. 70S ribosomes and 80S ribosomes; S’ stands for Svedberg’s units.  Prokaryotic bacteria and Archaeal cells contain 70S ribosomes, nearly 20,000 (30-35000) per cell, and surprisingly eukaryotic organisms also contain 70s like ribosomes in mitochondria and plastids, for these organelles are considered as symbionts of bacterial origin.  The organelle genomic RNA when compared to that of bacterial ribosomal RNAs, shows certain similarities in their sequence and structural organization.  Ribosomes are the most dynamic cellular machine with 2.5 MD mass with RNA accounting 2/3rds of the ribosomes.

 

http://www.biochem.umd.edu/

 

https://www.cancerwatch.org

 

Eukaryotes have 80s type of ribosomes, ten to twenty million per cell, are found in cytoplasm.  Each ribosome is actually made up of two subunits 60s and 40s and they can be separated into individual smaller subunits by lowering Mg2+ concentration and they can be sedimented into individual components.

 

When purified ribosomes from both sources are subjected to Phenol-chloroform extraction, one can obtain both RNAs and Riboproteins, which can be further purified to homogeneity; the following are the components of ribosomes.

 

 

 

 

 

Front and side views of ribosomal subunits; M. Oaks, A. Scheirman,

 http://www.pearsonhighered.com

 

 

Components of 70S Ribosomes:

 

 

 

 

 

 

 

 

 

Components of 80s ribosomes

 

 

                                   

70s

Size and ntds

No.Proteins

No. sites modified

50s

5s-120

 

23

 

23s-2900

34

 

30s

16s-1540

21

11

 

 

 

 

 

80s-ribosomes

 Size and ntds

No.Proteins

No. sites modified

40s

18s-1843-1900

33

43-44 2’O CH3 +~U

60s

28s-4710-4599

40

74,2’O-CH3, + ~U

5.8

160

 

nil

5s

120

 

 

 

 



 

 

 

Organelle Ribosomes:

Chloroplasts and mitochondria are semiautonomous structures.  They have their own heritable genetic material i.e. DNA; they have ribosomes and enzymes for performing specific functions.  Their genomes codes for 13 and ~120proteins (mit and Cp), two rRNAs and twenty two tRNA and rest of the required proteins are coded for by the nuclear genome.  Both chloroplast and mitochondrial ribosomes contain ~70s ribosomes similar to that of bacterial cells, there are some significant differences in their structural proteins and sizes.

 

 

 

Differences between chloroplast ribosomes left and bacterial ribosomes right; Manjuli R. Sharma et al; http://www.pnas.org/

 

 

Ribosomal RNA and Riboproteins:

 

 

Ribosomal subunit 3D models and rRNA in 3d organization; https://www.cancerwatch.org

 

 

 

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 provides space for the binding of ribosomal proteins to suggest overall structural features of ribosomes.

 

 

Riboproteins (Prokaryotic):

 

 

 

Two-dimensional Electrophoresis; In first dimension Isoelectric focusing or immobilized pH gradient Gel (IPG); followed by SDS-PAGE at 90o to that of first (2nd dimension). http://en.wikibooks.org/

 

http://www.nobelprize.org/

Gel Electrophoresis of Ribosomal Proteins;

Ribosomes contains mainly basic proteins (proteins that are positively charged at neutral pH). In addition the ribosome also contains some neutral or acidic proteins. The proteins can be extracted from the ribosome and separated by two-dimensional gel electrophoresis. These 2D-techniques usually separate proteins according to charge in the first dimension and according to size or charge (at a different pH) in the second dimension.Nobelprize.org

 

 

Figure 2

 

Two-dimensional polyacrylamide gel analysis of ribosomal proteins from wild-type and L27-lacking E. coli. Proteins were extracted from 10A 260 units of purified 70 S ribosomes from strains LG90 (left) and IW326 (right) and separated by acidic two-dimensional PAGE Iwona K. Wower, and Robert A. Zimmermann§

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Assembly:

 

 

http://micro.magnet.fsu.edu/

 

Ribosomes, tiny organelles composed of approximately 60 percent ribosomal RNA (rRNA) and 40 percent protein. However, though they are generally described as organelles, it is important to note that ribosomes are not bound by a membrane and are much smaller than other organelles. Some cell types may hold a few million ribosomes, but several thousand is more typical. The organelles require the use of an electron microscope to be visually detected; http://micro.magnet.fsu.edu/

Mloecular odel of tRNA bond to different ribosomal sites;E-site tRNA (1), P-site tRNA (2), A-site tRNA (3); http://www.chegg.com/

 

 

The ribbon diagram shows the positioning of tRNA on large ribosomal surface; A, amino acyl tRNA site, P peptidyl RNA site and E Exit site.

Laurence Moran; http://sandwalk.blogspot.in/

 

 

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.

 

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

 

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]

           

James R. Williamson et al ; http://www.scripps.edu/

 

Assembly reaction of the 30S ribosomal subunit. A total of 20 proteins (represented as circles) bind to the 16S ribosomal RNA (shown as a line). In the absence of the proteins, the RNA is only partly folded (left), but it becomes highly ordered as it folds during assembly. The complex reaction shown here occurs in a stepwise manner to ensure proper assembly, but the details of this process remain to be elucidated.Williamson

 

Ribosome Biogenesis and the Translation Process in Escherichia coli;

http://mmbr.asm.org/content

Assembly of 30s ribosomal protein on 16srRNA; JOEL F. GRONDEK and GLORIA M. CULVER; http://rnajournal.cshlp.org/

 

Studies of Escherichia coli 30S ribosomal subunit assembly have revealed a hierarchical and cooperative association of ribosomal proteins with 16S ribosomal RNA; these results have been used to compile an in vitro 30S subunit assembly map. In single protein addition and omission studies, ribosomal protein S13 was shown to be dependent on the prior association of ribosomal protein S20 for binding to the ribonucleoprotein particle. While the overwhelming majority of interactions revealed in the assembly map are consistent with additional data, the dependency of S13 on S20 is not. Structural studies position S13 in the head of the 30S subunit > 100 Å away from S20, which resides near the bottom of the body of the 30S subunit. All of the proteins that reside in the head of the 30S subunit, except S13, have been shown to be part of the S7 assembly branch, that is, they all depend on S7 for association with the assembling 30S subunit. Given these observations, the assembly requirements for S13 were investigated using base-specific chemical footprinting and primer extension analysis. These studies reveal that S13 can bind to 16S rRNA in the presence of S7, but not S20. Additionally, interaction between S13 and other members of the S7 assembly branch have been observed. These results link S13 to the 3′ major domain family of proteins, and the S7 assembly branch, placing S13 in a new location in the 30S subunit assembly map where its position is in accordance with much biochemical and structural data. JOEL F. GRONDEK and GLORIA M. CULVER

 

                                                                                   

 

 

 

 

Role of rRNA in protein synthesis (Prokaryotic):

 

 

 

 

 

 

 

 

 

 

 

Structural Features (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 contains 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 20 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.

 

 

 

 

Protein exit tunnel in large ribosomal subunit; A. A. Bogdanov*, N. V. Sumbatyan et al;;http://protein.bio.msu.ru/

 

 

 

 

 

 

 

 

 

 

This diagram shows a tunnel through which the nascent polypeptide threads through as it is translated. Interplay between the Ribosomal Tunnel, Nascent Chain;Agata L. Starosta et al; http://www.cipsm.de/

 

 

 

 

 

Signal sequence dependent SRP-Ribosome interaction; Binding/association of SRP protein at exit tunnel and guide the nascent protein into ER receptor and channel; http://edoc.hu-berlin.de/

 

Model for the first steps of SRP cycle;  SRP binds to protruding Hydrophopic signal sequences and guides towards ER channel; http://edoc.hu-berlin.de/

 

 

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 at 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. 

 

 

 

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.