RNA structure and properties

The Structure of RNA

• RNA, like DNA, is a polymer consisting of nucleotides joined together by phosphodiester bonds.

• Whereas DNA nucleotides contain deoxyribose sugars, RNA nucleotides have ribose sugars.

• With a free hydroxyl group on the 2-carbon atom of the ribose sugar, RNA is degraded rapidly under alkaline conditions

• The deoxyribose sugar of DNA lacks this free hydroxyl group; so DNA is a more stable molecule.

• Another important difference is that thymine, one of the two pyrimidines found in DNA, is replaced by uracil in RNA.

• RNA is usually single stranded, consisting of a single polynucleotide strand.

• Although RNA is usually single stranded, short complementary regions within a nucleotide strand can pair and form secondary structures.

• These RNA secondary structures are often called hairpin-loops or stem-loop structures.

• When two regions within a single RNA molecule pair up, the strands in those regions must be antiparallel, with pairing between cytosine and guanine and between adenine and uracil.

The Structure of Messenger RNA

Shine-Dalgarno sequence

• The Shine-Dalgarno sequence (or Shine-Dalgarno box), is a ribosomal binding site in the mRNA, generally located 8 basepairs upstream of the start codon AUG.

• The Shine-Dalgarno sequence exists only in prokaryotes. • The six-base consensus sequence is AGGAGG;

– in E. coli, for example, the sequence is AGGAGGU. • This sequence helps recruit the ribosome to the mRNA to

initiate protein synthesis by aligning it with the start codon.• The complementary sequence (UCCUCC), is called the anti-

Shine-Dalgarno sequence and is located at the 3' end of the 16S rRNA in the ribosome.

• The eukaryotic equivalent of the Shine-Dalgarno sequence is called the Kozak sequence. – (GCC)RCCATGG where R is a purine (A or G) three bases upstream

of the start codon (AUG), which is followed by another 'G'.

Poly(A) Tail

• Most mature eukaryotic mRNAs have from 50 to 250 adenine nucleotides at the 3 end (a poly(A) tail).

• The poly(A) tail confers stability on many mRNAs, increasing the time during which the mRNA remains intact and available for translation before it is degraded by cellular enzymes.

• The stability conferred by the poly(A) tail is dependent on the proteins that attached to the tail– 3 to 5% of the total cellular RNA.

Ribosomal RNA

• 80% of the total RNA of the cell. • Ribosomal RNA consists of a single strand twisted

upon itself in some regions. • It has helical regions connected by intervening single

strand regions. • The helical regions may show presence or absence of

positive interaction. • In the helical region most of the base pairs are

complementary, and are joined by hydrogen bonds. • In the unfolded single strand regions the bases have

no complements.

RNA secondary Structure representation

Large subunitsrRNA

Small subunitrRNA

Transfer RNA

• The second most common RNA in the cell is transfer RNA.

• It is also called soluble RNA because it is too small to be precipitated by ultracentrifugation at 100,000 g.

• It constitutes about 10-20% of the total RNA of the cell.

• Transfer RNA is a relatively small RNA having a molecular weight of about 25,000 to 30,000 and the sedimentation coefficient of mature eukaryote tRNA is 3.8S.

tRNA

• The nucleotide sequence (primary structure) of tRNA was first worked out by Holley et al (1965) for yeast alanine tRNA.

• Most tRNAs contain between 74 and 95 nucleotides, some of which are complementary to each other and form intramolecular hydrogen bonds.

• As a result, each tRNA has a cloverleaf structure• The four major arms are the acceptor arm, the TψC

arm, the anticodon arm, and the DHU arm• Most cells have 40 to 50 distinct tRNAs, and

eukaryotic cells have multiple copies of many of the tRNA genes.

Acceptor Arm• The acceptor stem consists of 7 base pairs and 4

unpaired nucleotide units. • The latter include a constant 3' terminal -CCA

sequence and a fourth nucleotide which is a variable purine (A or G).

• The amino acid molecule attaches to the 3'OH terminal of the -CCA sequence, which is' known as the amino acid binding site.

• The 5' end of tRNA is either guanine (G) or cytosine (C)

• It has a 3' terminal site for amino acid attachment. This covalent linkage is catalyzed by an aminoacyl tRNA synthetase.

D arm

• It consists of 15-18 nucleotides with 3-4 base pairs in the stem and 7-11 unpaired nucleotides in the loop.

• The loop of the D arm is called Loop I or dihydrouridine (DHU) loop or the D loop.

• These regions consist of 1-3 nucleotides, mostly pyrimidines, with a high proportion of DHU.

• The synthetase site which recognizes the amino acid activating enzyme is located on a part of the D loop and a part of the acceptor stem on the 5' side.

Anticodon arm

• The third arm or anticodon arm consists of an anticodon stem of 5 base pairs and a loop, called Loop II or the anticodon loop.

• This loop consists of 7 unpaired nucleotides of which the middle three form the anticodon.

• The anticodon recognises the 3 complementary bases which constitute the codon of mRNA.

• On the 3' side of the anticodon is a hypermodified purine (H) while on the 5' side is U and a pyrimidine (Y).

TψC arm• The TψC arm consists of a stem having 5 base pairs

and a loop of 7 nucleotides. • The outermost of the 5 pairs of the stem is C-G. • The TψC loop contains a constant TψC sequence so

named because of presence of ribothymidine [T], pseudouridine[Ψ] and cytidine [C] residues

• All tRNAs have a ribosome recognition site on the TψC loop consisting of a G-T-ψ-C-R sequence.

• The Tψ arm is involved in the binding of tRNA molecules to molecules to the ribosomes.

• The variable arm (the "lump", the miniloop, Loop III, extra arm) lies between the TψC arm and anticodon arm.

• Class I tRNA – Small variable loop containing 3-5 bases but no

stem. Represent 75% of all tRNAs.• Class II tRNA

– Large variable loop with 13-21 bases along with 5 base pairs in the variable stem

Variable arm

Invariant and Semi invariant Nucleotides

• When the sequences of tRNAs are compared, the bases found at some positions are invariant (or conserved); almost always a particular base is found at the position.

• Some positions are described as semi invariant (or semi conserved) because they are restricted to one type of base (purine versus pyrimidine), but either base of that type may be present.

Unusual Bases in tRNA

• Precursor tRNA molecules transcribed on the DNA template contains the usual bases. These are then modified to unusual bases.

• The unusual bases are important because they protect the tRNA molecule against degradation by RNase.

• Some of the unusual bases of tRNA are methyl guanine (GMe), dimethylguanine(GMe2), methylcytosine (Me), ribothymine (T), pseudouridine (ψ), dihydrouridine (DHU), inosine (I) and methylinosine (IMe, MeI).

non-Watson-Crick associations

• Within a given tRNA, most of the base pairings are conventional partnerships of A-U and G-C, but occasional G-U, G-ψ, or A-ψ pairs are found. The additional types of base pairs are less stable than the regular pairs, but still allow a double-helical structure to form in RNA.

• The four main wobble base pairs are guanine-uracil, inosine-uracil, inosine-adenine, and inosine-cytosine (G-U, I-U, I-A and I-C)

Charging of tRNAs

• When a tRNA is charged with the amino acid corresponding to its anticodon, it is called aminoacyl-tRNA. (tRNATyr)

• The amino acid is linked by an ester bond from its carboxyl group to the 2' or 3' hydroxyl group of the ribose of the 3' terminal base of the tRNA (which is always adenine).

• The process of charging a tRNA is catalyzed by a specific enzyme, aminoacyl-tRNA synthetase.

• There are (at least) 20 aminoacyl tRNA synthetases. • Each recognizes a single amino acid and all the

tRNAs on to which it can legitimately be placed. • If there is more than one tRNA for the same amino

acid, subscript numerals are used to distinguish them. So two tRNAs for tyrosine would be described as tRNAl Tyr and tRNA2

Tyr

• Isoaccepting tRNA that carries same amino acids.

• charged - tRNA with amino acid attached uncharged - tRNA, no amio acid mischarged - incorrect amino acid

Tertiary Structure of tRNA

• The secondary structure of each tRNA folds into a compact L-shaped tertiary structure in which the 3' end that binds the amino acid is distant from the anticodon that binds the mRNA.

• The base paired double-helical stems of the secondary structure are maintained in the tertiary structure, but their arrangement in three dimensions essentially creates two double helices at right angles to each other.

• The acceptor stem and the TψC C stem form one continuous double helix with a single gap; the D stem and anticodon stem form another continuous double helix, also with a gap.

• The region between the double helices, where the turn in the L-shape is made, contains the TψC loop and the D loop.

• So the amino acid resides at the extremity of one arm of the L-shape and the anticodon loop forms the other end which is consistent with their roles in protein synthesis.

Tertiary Structure of tRNA

• In that year, the 2.5-Å resolution X-ray crystal structure of yeast tRNAPhe was separately elucidated by Alexander Rich in collaboration with Sung Hou Kim and, in a different crystal form, by Aaron Klug.

• The molecule assumes an L-shaped conformation in which one leg of the L is formed by the acceptor and T stems folded into a continuous A-RNA-like double helix and the other leg is similarly composed of the D and anticodon stems

• Each leg of the L is 60 Å long and the anticodon and amino acid acceptor sites are at opposite ends of the molecule, some 76 Å apart.

• The narrow 20- to 25-Å width of native tRNA is essential to its biological function:

Classification of tRNA

• A Study of different tRNAs shows that the structure of the acceptor stem, the anticodon arm and the TψC arm are constant.

• The differences in the tRNAs lie in the D arm and the variable arm.

• Based on the differences in these two variable regions, three classes of tRNA have been recognized.– Class I (D4-V4-5), with 4 base pairs in the D stem and 4-5 bases in the

variable loop.

– Class II (DS-V4-5), with 3 base pairs in the D stem and 4-5 bases pairs in the variable loop.

– Class III (D3-VN), with 3 base pairs in the D stem and a large variable arm.

3D Structure of RNA

• The product of transcription of DNA is always single-stranded RNA.

• The single strand tends to assume a right-handed helical conformation dominated by base stacking interactions which are stronger between two purines than between a purine and pyrimidine or between two pyrimidines.

• The purine-purine interaction is so strong that a pyrimidine separating two purines is often displaced from the stacking pattern so that the purines can interact.

• Any self-complementary sequences in the molecule produce more complex structures.

• RNA can base-pair with complementary regions of either RNA or DNA.

• Base pairing matches the pattern for DNA:

• G pairs with C and • A pairs with U • One difference is that base pairing

between G and U residues—unusual in DNA—is fairly common in RNA

• The paired strands in RNA or RNA-DNA duplexes are antiparallel, as in DNA.

• Where complementary sequences are present, the predominant double-stranded structure is an A-form right-handed double helix.

• A-RNA or RNA-11,– 11bp per turn – pitch of 30.9 Å– base pairs inclined to the helix axis by 16.7.

• Z-form helices have been made in the laboratory (under very high-salt or high-temperature conditions).

• The B form of RNA has not been observed.

Some RNAs Are Enzymes

• Until about 20 years ago, all known enzymes were proteins. But then it was discovered that some RNA molecules can act as enzymes; that is, catalyze covalent changes in the structure of substrates (most of which are also RNA molecules).

• Catalytic RNA molecules are called ribozymes.

• Ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA

• Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds, or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome.

• 1982:Self-splicing in protozoan Tetrahymena pre-rRNA (group I intron)

• One of the first ribozymes to be discovered was RNAse P, a ribonuclease that is involved in generating tRNA molecules from larger, precursor RNAs.

• RNAse P is composed of both RNA and protein; however, the RNA moiety alone is the catalyst.