Microbiology and Immunology

WORLD OF MICROBIOLOGY AND IMMUNOLOGY Ribonucleic acid (RNA)

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The mRNA molecules contain at the 5’ end a leader

sequence that is not translated, known as UTR (untranslated

region) and an initiation codon (AUG), that precedes the cod-

ing region formed by the spliced exons, which are termed

codons in the mature mRNA. At the end of the coding region,

three termination codons (UAG, UAA, UGA) are present,

being followed by a trailer sequence that constitutes another

UTR, which is by its turn followed by the poly-A tail. The sta-

bility of the mRNA molecule is crucial to the proper transla-

tion of the transcript into protein. The poly-A tail is

responsible by such stability because it prevents the preco-

cious degradation of mRNA by a 3’ to 5’ exonuclease (a cyto-

plasmatic enzyme that digests mRNA starting from the

extremity 3’ when the molecule leaves the cell nucleus). The

mRNA of histones, the nuclear proteins that form the nucleo-

somes, do not have poly-A tails, thus constituting an exception

to this rule. The poly-A tail also protects the other extremity of

the mRNA molecule by looping around and touching the 7-

methylguanosine cap attached to the 5’ extremity. This pre-

vents the decapping of the mRNA molecule by another

exonuclease. The removal of the 7-methylguanosine exposes

the 5’ end of the mRNA to digestion by the 5’ to 3’ exonucle-

ase (a cytoplasmatic enzyme that digests mRNA starting from

the 5’ end). When the translation of the protein is completed,

the enzymatic process of deadenylation (i.e., enzymatic diges-

tion of the poly-A tail) is activated, thus allowing the subse-

quent mRNA degradation by the two above mentioned

exonucleases, each working at one of the ends of the molecule.

Transfer RNA (tRNA) is often referred to as the

“Rosetta Stone” of genetics, as it translates the instructions

encoded by DNA, by way of messenger RNA (mRNA), into

specific sequences of amino acids that form proteins and

polypeptides. This class of small globular RNA is only 75 to

90 nucleotides long, and there is at least one tRNA for every

amino acid. The job of tRNA is to transport free amino acids

within the cell and attach them to the growing polypeptide

chain. First, an amino acid molecule is attached to its particu-

lar tRNA. This process is catalyzed by an enzyme called

aminoacyl—tRNA synthetase that binds to the inside of the

tRNA molecule. The molecule is now charged. The next step,

joining the amino acid to the polypeptide chain, is carried out

inside the ribosome. Each amino acid is specified by a partic-

ular sequence of three nucleotide bases called codons. There

are four different kinds of nucleotides in mRNA. This makes

possible 64 different codons (4^3 ). Two of these codons are

called STOP codons; one of these is the START codon (AUG).

With only 20 different amino acids, it is clear that some amino

acids have more then one codon. This is referred to as the

degeneracy of the genetic code. On the other end of the tRNA

molecule are three special nucleotide bases called the anti-

codon. These interact with three complimentary codon bases

in the mRNA by way of hydrogen bonds. These weak direc-

tional bonds are also the force that holds together the double

strands of DNA.

In order to understand how this happens, it was neces-

sary to first understand the three dimensional structure (con-

formation) of the tRNA molecule. This was first attempted in

1965, where the two-dimensional folding pattern was deduced

from the sequence of nucleotides found in yeastalanine tRNA.

Later work (1974), using x-ray diffraction analysis, was able to

reveal the conformation of yeast phenylalanine tRNA. The

molecule is shaped like an upside-down L. The vertical portion

is made up of the D stem and the anti-codon stem, and the hor-

izontal arm of the L is made up of the acceptor stem and the T

stem. Thus, the translation depends entirely upon the physical

structure. At one end of each tRNA is a structure that recog-

nizes the genetic code, and at the other end is the particular

amino acid for that code. Amazingly, this unusual shape is con-

served between bacteria, plants, and animals.

Another unusual thing about tRNA is that it contains

some unusual bases. The other classes of nucleic acids can

undergo the simple modification of adding a methyl (CH3–)

group. However, tRNA is unique in that it undergoes a range

of modifications from methylation to total restructuring of the

purine ring. These modifications occur in all parts of the tRNA

molecule, and increase its structural integrity and versatility.

Ribosomes are composed of ribosomal RNA (as much

as 50%) and special proteins called ribonucleoproteins. In

eukaryotes(an organism whose cells have chromosomes with

nucleosomal structure and are separated from the cytoplasm

by a two membrane nuclear envelope and whose functions are

compartmentalized into distinct cytoplasmic organelles), there

are actually four different types of rRNA. One of these mole-

cules is called 18SrRNA; along with some 30–plus different

proteins, it makes up the small subunit of the ribosome. The

other three types of rRNA are called 28S, 5.8S, and 5S rRNA.

One of each of these molecules, along with some 45 different

proteins, is used to make the large subunit of the ribosome.

There are also two rRNAs exclusive to the mitochondrial (a

circular molecule of some 16,569 base pairs in the human)

genome. These are called 12S and 16S. A mutation in the

12SrRNA has been implicated in non-syndromic hearing loss.

Ribosomal RNA’s have these names because of their molecu-

lar weight. When rRNA is spun down by ultracentrifuge, these

molecules sediment out at different rates because they have

different weights. The larger the number, the larger the

molecule.

The larger subunit appears to be mainly involved in

such biochemical processes as catalyzing the reactions of

polypeptide chain elongation and has two major binding sites.

Binding sites are those parts of large molecule that actively

participate in its specific combination with another molecule.

One is called the aminoacyl site and the other is called the pep-

tidyl site. Ribosomes attach their peptidyl sites to the mem-

brane surface of the rER. The aminoacyl site has been

associated with binding transfer RNA. The smaller subunit

appears to be concerned with ribosomal recognition processes

such as mRNA. It is involved with the binding of tRNA also.

The smaller subunit combines with mRNA and the first

“charged “ tRNA to form the initiation complex for translation

of the RNA sequence into the final polypeptide.

The precursor of the 28S, 18S and the 5.83S molecules

are transcribed by RNA polymerase I (Pol I) and the 5S rRNA

is transcribed by RNA polymerase III (PoIII). Pol I is the most

active of all the RNA polynmerases, and is one indication of

how important these structures are to cellular function.

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