Ribosomal RNA

Filed under: by: Varadharajan


The ribosome is a large machinery (~ 20 nm in diameter, 70S sedimentation rate for bacterial ribosomes) and is made of two subunits: a large subunit (~50S) and a small subunit (~ 30S). The large subunit is in turn made of two ribosomal RNA (5S and 23S) and several (~34 proteins) whereas the small subunit has one ribosomal RNA (16S) and ~ 21 proteins. The 23S rRNA is ~ 3000 nucleotides long, and the 16S rRNA is ~ 1500 nucleotides long.

The structures of ribosomal RNA can get very complicated because of the large number of ways in which hairpins and loops can be formed. Predicting these structures requires a combination of both computational methods (in which the most probable secondary structures are determined from estimates of free energy for a given structure) and a variety of experimental techniques.

Oligonucleotide mapping techniques

This technique is useful in identifying exposed single-stranded regions of a folded RNA molecule by hybridization with short synthesized nucleotide chains (also called oligonucleotides) that are complementary to, for instance, the loop regions in RNA.

Folded RNA molecules are confined to one region in space separated by another region by a semi-permeable membrane. On the other side of the partition are radioactive oligonucleotides (~ 5-10 nucleotides long) that can pass through the membrane and bind to RNA molecules, but the RNA molecules, which are much bigger in size, cannot.

At equilibrium, free oligomers are in the same concentration on both sides of the partition. However, the radioactivity on the side with the RNA molecules is larger than the other size because some oligomers will associate with (bind to) RNA if the sequences of oligomers and loop regions are complementary. A measure of the ratio (rd) of radioactivity from either side gives a measure of the binding or association constant where [X] is the concentration of the RNA-oligomer complex, [O] is the free oligomer concentration on either side, and [RNA] is the concentration of molecules that are not bound to an oligomer.

The ratio

If [RNA] >> [O], then the RNA concentration can be assumed the same before and after mixing and the ratio becomes .

Therefore, a measurement of rd yields a direct measure of Ka.

All oilgonucleotides will lead to some association since there is always a match at a single base-pair level. Therefore for any oligonucleotide. For oligonucleotides ~ 4 bases long that match an exposed loop region on the RNA the free energy change upon association is substantially larger (by ~ 10-15 kBT ) than the free energy change from single base-pair matches. This lead to an increase in the association constant by a factor of 104 to 106.

This technique can easily distinguish between two possible conformations of an RNA molecule which have different sequences in their loop regions.


We can also estimate which structure is more probable (i.e. which one has the lower free energy.

The free energy of a hairpin can be broken into two parts, the free energy of forming a loop closed by a single base-pair and the free energy for the base-paired `stem' of the hairpin.

In RNA molecules the most probable loop size consists of ~ 6-7 bases in the loop. Smaller loops are energetically unfavorable as a result of steric hindrances among the bases and atoms of the backbone. Larger loops are entropically unfavorable. The loss of entropy when loops are formed increases with increasing loop size.

for the optimal sized loop closed by a G-C base-pair is ~ 7-8 kBT in 1M NaCl. In our example we have a loop with 10 bases in structure 1 () and 2 loops with 4 bases each in structure 2 (for each loop).

Note that is a positive quantity; it is unfavorable to make loops relative to the random coil conformation.

The hairpin structures are stabilized when the free energy gain from base-pair formation exceeds the free energy cost of loop formation.

The gain from adding a base-pair to an already existing G-C pair is ~ for adding a G-C base-pair and ~ for adding a A-U base-pair.

Therefore the net change in free energy for structure 1 is

and for structure 2 is

Structure 1 is more stable (although marginally) and the relative populations of the two structures are given by the Boltzmann distribution

comments (1)

Secondary and tertiary structures of tRNA molecules

Filed under: by: Varadharajan


The crystal structures of several tRNA molecules have been determined. All tRNA molecules have very similar secondary structures in which the single-stranded chain is folded in a 'clover-leaf' structure that has three hairpins and an acceptor stem where the amino-acid is covalently attached. The acceptor stem is the 3' end of the chain and always terminates in the sequence 5'-CCA-3'.

This particular tRNA is specific for the amino-acid Alanine whose codon on the mRNA is 5'-GCC-3' and the anti-codon loop of tRNA reads 5'-GGC-3'. The grey circles are examples of unusual, chemically modified, bases.

The secondary structure then folds up to form a 3-dimensional structure which looks like an inverted L.

One end of one L arm (the 3' end of the chain) is the acceptor stem. The other end of the L is the anti-codon loop that has to match the codon on the mRNA. The distance between the two ends of the L is ~ 7 nm. The corner of the L is used for correct positioning on the ribosome where the protein synthesis takes place.

In the tertiary (3-dimensional) structures of RNA, bases sometimes make hydrogen bonds with more than one partner, as illustrated in the picture above. These extra hydrogen bonds compensate for the distortion in the double-stranded helical regions when the RNA folds up and help stabilize the tertiary structure.

The covalent attachment between the tRNA and its corresponding amino-acid is achieved by yet another adaptor molecule (this time a protein molecule called aminoacyl-tRNA synthetase) of which there are at least 20 varieties, one for each kind of amino-acid. The synthetases recognize the detailed shape and properties of a specific amino-acid and the detailed shape of the acceptor stem in the folded tRNA molecule and catalyze the covalent attachment between the amino-acid and its corresponding tRNA.

comments (0)

RNA structures

Filed under: by: Varadharajan

RNA molecules are also polynucleotides with a sugar-phosphate backbone and four kinds of bases. The main differences between RNA and DNA are:

  • RNA molecules are single-stranded
  • The sugar in RNA is a ribose sugar (as opposed to deoxy-ribose) and has an –OH at the 2' C position highlighted in red in the figure below (DNA sugars have –H at that position)
  • Thymine in DNA is replaced by Uracil in RNA. T has a methyl (-CH3) group instead of the H atom shown in red in U.

The picture shows an ATP molecule (adenosine tri-phosphate) about to be incorporated into an RNA chain with the release of a di-phosphate).

RNA molecules do not have a regular helical structure like DNA. Instead, they can form complicated 3-dimensional structures where the strands can loop back and form intra-strand base-pairs from self-complementary regions along the chain.

comments (0)

Ribonucleic acid

Filed under: by: Varadharajan

Ribonucleic acid (RNA) is a biologically important type of molecule that consists of a long chain of nucleotide units. Each nucleotide consists of a nitrogenous base, a ribose sugar, and a phosphate.
RNA is very similar to DNA, but differs in a few important structural details: in the cell, RNA is usually single-stranded, while DNA is usually double-stranded; RNA nucleotides contain ribose while DNA contains deoxyribose (a type of ribose that lacks one oxygen atom); and RNA has the base uracil rather than thymine that is present in DNA. RNA is transcribed from DNA by enzymes called RNA polymerases and is generally further processed by other enzymes. RNA is central to protein synthesis. Here, a type of RNA called messenger RNA carries information from DNA to structures called ribosomes. These ribosomes are made from proteins and ribosomal RNAs, which come together to form a molecular machine that can read messenger RNAs and translate the information they carry into proteins. There are many RNAs with other roles – in particular regulating which genes are expressed, but also as the genomes of most viruses.

comments (0)

Comparison with DNA

Filed under: by: Varadharajan

RNA and DNA are both nucleic acids, but differ in three main ways. First, unlike DNA which is double-stranded, RNA is a single-stranded molecule in most of its biological roles and has a much shorter chain of nucleotides. Second, while DNA contains deoxyribose, RNA contains ribose (there is no hydroxyl group attached to the pentose ring in the 2' position in DNA). These hydroxyl groups make RNA less stable than DNA because it is more prone to hydrolysis. Third, the complementary base to adenine is not thymine, as it is in DNA, but rather uracil, which is an unmethylated form of thymine.

comments (0)

Messenger RNA

Filed under: by: Varadharajan

Messenger RNA (mRNA) is the RNA that carries information from DNA to the ribosome, the sites of protein synthesis (translation) in the cell. The coding sequence of the mRNA determines the amino acid sequence in the protein that is produced.[20] Many RNAs do not code for protein however (about 97% of the transcriptional output is non-protein-coding in eukaryotes.
These so-called non-coding RNAs ("ncRNA") can be encoded by their own genes (RNA genes), but can also derive from mRNA introns.[25] The most prominent examples of non-coding RNAs are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation.[1] There are also non-coding RNAs involved in gene regulation, RNA processing and other roles. Certain RNAs are able to catalyse chemical reactions such as cutting and ligating other RNA molecules,[26] and the catalysis of peptide bond formation in the ribosome;[3] these are known as ribozymes.
In translation
Messenger RNA (mRNA) carries information about a protein sequence to the ribosomes, the protein synthesis factories in the cell. It is coded so that every three nucleotides (a codon) correspond to one amino acid. In eukaryotic cells, once precursor mRNA (pre-mRNA) has been transcribed from DNA, it is processed to mature mRNA. This removes its introns—non-coding sections of the pre-mRNA. The mRNA is then exported from the nucleus to the cytoplasm, where it is bound to ribosomes and translated into its corresponding protein form with the help of tRNA. In prokaryotic cells, which do not have nucleus and cytoplasm compartments, mRNA can bind to ribosomes while it is being transcribed from DNA. After a certain amount of time the message degrades into its component nucleotides with the assistance of ribonucleases.[20]
Transfer RNA (tRNA) is a small RNA chain of about 80 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino acid attachment and an anticodon region for codon recognition that binds to a specific sequence on the messenger RNA chain through hydrogen bonding.[25]
Ribosomal RNA (rRNA) is the catalytic component of the ribosomes. Eukaryotic ribosomes contain four different rRNA molecules: 18S, 5.8S, 28S and 5S rRNA. Three of the rRNA molecules are synthesized in the nucleolus, and one is synthesized elsewhere. In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein synthesis. Several ribosomes may be attached to a single mRNA at any time.[20] rRNA is extremely abundant and makes up 80% of the 10 mg/ml RNA found in a typical eukaryotic cytoplasm.[27]
Transfer-messenger RNA (tmRNA) is found in many bacteria and plastids. It tags proteins encoded by mRNAs that lack stop codons for degradation and prevents the ribosome from stalling.[28]

comments (0)

Subscribe to: Posts (Atom)