PPRROOCCEESSSSIINNGG OOFF ttRRNNAA

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ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA

P P R R O O C C E E S S S S I I N N G G O O F F t t R R N N A A

B B B Y Y Y

D D R R . . P P O O U U L L A A M M I I A A D D H H I I K K A A R R Y Y M M U U K K H H E E R R J J E E E E A A S S S S I I S S T T A A N N T T P P R R O O F F E E S S S S O O R R

D D E E P P A A R R T T M M E E N N T T O O F F Z Z O O O O L L O O G G Y Y

N N A A R R A A J J O O L L E E R R A A J J C C O O L L L L E E G G E E

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ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA

tRNA undergoes extensive processing. The mature tRNAs consists of 80-90 nucleotides. But the precursor tRNA is much longer.

For example tRNA

^Tyr

which is a tyrosine carrying tRNA

contains 350 nucleotides. Processing discards useless

sequences. This is done by enzymes RNase D, RNase E,

RNase F and RNase P. Nucleotides are removed from both

5′ and 3′ ends.

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Endonucleases also remove many sequences. Cleaving is done after the primary transcript has folded and formed characteristic stems and loop structure by extensive complementary base pairing. RNase P is a ribozyme.

The 5′-CCA-3′ sequence at 3′-end of mature tRNA is added

by the enzyme tRNA nucleotidyl transferse. This generates

the mature 3′-end the tRNA.

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Several unusual bases are formed by the modification of normal existing bases A, G, C and U by the enzymatic action.

These modified bases are pseudouridine (Ψ), 2-

isopentenyladenosine (2 ip A), 2-O-methylguanosine (2m

G), 4-thiouridine (4 t μ), Ribothymidine, dihyrouridine and

inosine.

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Each different tRNA binds to a specific amino acid and transfers it to the ribosome.

Mature tRNAs take on a three-dimensional structure

through intramolecular basepairing to position the amino

acid binding site at one end and the anticodon in an

unbasepaired loop of nucleotides at the other end.

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The anticodon is a three-nucleotide sequence, unique to each different tRNA, that interacts with a messenger RNA (mRNA) codon through complementary base pairing.

There are different tRNAs for the 21 different amino acids.

Most amino acids can be carried by more than one tRNA.

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.

Figure: Structure of tRNA: This is a space-filling model of a tRNA molecule that adds the amino acid phenylalanine to a growing polypeptide chain. The anticodon AAG binds the codon UUC on

the mRNA. The amino acid phenylalanine is attached to the other end of the tRNA.

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ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA

This is a space-filling model of a tRNA molecule that adds the amino acid phenylalanine to a growing polypeptide chain.

The anticodon AAG binds the codon UUC on the mRNA.

The amino acid phenylalanine is attached to the other end

of the tRNA.

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ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA

In all organisms, tRNAs are transcribed in a pre-tRNA form that requires multiple processing steps before the mature tRNA is ready for use in translation.

In bacteria, multiple tRNAs are often transcribed as a single RNA.

The first step in their processing is the digestion of the

RNA to release individual pre-tRNAs. In archaea and

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ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA

eukaryotes, each pre-tRNA is transcribed as a separate transcript.

The processing to convert the pre-tRNA to a mature tRNA involves five steps.

1. The 5′ end of the pre-tRNA, called the 5′ leader sequence, is cleaved off.

2. The 3′ end of the pre-tRNA is cleaved off.

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ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA

3. In all eukaryote pre-tRNAs, but in only some bacterial and archaeal pre-tRNAs, a CCA sequence of nucleotides is added to the 3′ end of the pre-tRNA after the original 3′

end is trimmed off. Some bacteria and archaea pre-tRNAs

already have the CCA encoded in their transcript

immediately upstream of the 3′ cleavage site, so they don’t

need to add one. The CCA at the 3′ end of the mature tRNA

will be the site at which the tRNA’s amino acid will be

added.

(13)

4. Multiple nucleotides in the pre-tRNA are chemically modified, altering their nitrogen bases. On average about 12 nucleotides are modified per tRNA. The most common modifications are the conversion of adenine (A) to pseudouridine (ψ), the conversion of adenine to inosine (I), and the conversion of uridine to dihydrouridine (D).

But over 100 other modifications can occur.

(14)

5. A significant number of eukaryotic and archaeal pre- tRNAs have introns that have to be spliced out. Introns are rare in bacterial pre-tRNAs, but do occur occasionally and are spliced out.

After processing, the mature pre-tRNA is ready to have its

cognate amino acid attached.

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ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA

The cognate amino acid for a tRNA is the one specified by its anticodon.

Attaching this amino acid is called charging the tRNA.

In eukaryotes, the mature tRNA is generated in the

nucleus, and then exported to the cytoplasm for charging.

(16)

ZOOLOGY: SEM- V, PAPER- C11T: MOLECULAR BIOLOGY, UNIT 5: POST TRANSCRIPTIONAL MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA

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T T H H A A N N K K Y Y O O U U