tRNA (Transfer RNA) is a small RNA (Ribonucleic Acid) chain (74-93 nucleotides) that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has a site for amino acid attachment and a three-base region called the anticodon that recognizes the corresponding three-base codon region on mRNA via complementary base pairing. Each type of tRNA molecule can be attached to only one type of amino acid, but because the genetic code is degenerate - that is, it contains multiple codons that specify the same amino acid - multiple types of tRNA molecules bearing different anticodons may carry the same amino acid. tRNA has primary structure (the order of nucleotides from 5 to 3), secondary structure (usually visualized as the cloverleaf structure), and tertiary structure (all tRNAs have a similar L-shaped 3D structure that allows them to fit into the P and A sites of the ribosome) (Ref.1).
tRNA consist of a 5-terminal phosphate group, an acceptor stem (also called the amino acid stem), which is a 7-bp stem that includes the 5-terminal nucleotide and the 3-terminal nucleotide with the 3-terminal OH group (which is used to attach the amino acid). The acceptor stem may contain non-Watson-Crick base pairs. tRNA also contains the CCA tail, which is a CCA sequence at the 3 end of the tRNA molecule. This sequence is important for the recognition of tRNA by enzymes critical in translation. In eukaryotes, the CCA sequence is added during processing and therefore does not appear in the tRNA gene. Besides, it also contains a D arm, an Anticodon arm and a T arm. D arm is a 4 bp stem ending in a loop that often contains dihydrouridine. The Anticodon arm is a 5-bp stem whose loop contains the anticodon. The T arm is a 5 bp stem containing the sequence TØC where Ø is a pseudouridine. Bases that have been modified, especially by methylation, occur in several positions outside the anticodon. The first anticodon base is sometimes modified to inosine (derived from adenine) or pseudouridine (derived from uracil). Some tRNAs also include a thymine, a ribonucleotide that is typically not found in RNAs. There are multiple tRNA genes in eukaryotes. In the human genome, there are about 2000 non-coding RNA genes, which include tRNA genes. There are 22 mitochondrial tRNA genes; 497 nuclear genes encoding cytoplasmic tRNA molecules and there are 324 tRNA-derived putative pseudogenes. tRNAs are transcribed by RNA Pol-III
(RNA Polymerase III) (Ref.2, 3 & 4).
tRNA gene promoters consist of 2 separated 10bp elements (Box A and B) located downstream of the transcription start site. TFIIIC
(General Transcription Factor-IIIC) binds to Box B. TFIIIC
is composed of six polypeptides with a total molecular weight of 600 kDa. After binding to TFIIIC
, Box A orients TFIIIC
towards the start site. TFIIIC
causes correct positioning of TFIIIB
(Pre-Initiation Complex), which then recruits RNA Pol-III
. This DNA/TFIIIB
(General Transcription Factor-IIIB) /Pol III
initiation Complex is very stable and may pass through many rounds of tRNA transcription. TFIIIB
is made up of three subunits. One is TBP
(TATA-Binding Protein), which is a subunit of a general initiation factor for all three nuclear RNA Polymerases
. The second, called BRF
(TFIIB-Related Factor) is similar in sequence to TFIIB
, and performs a similar function in initiation by RNA Polymerase-III
does for TFIIB
does for RNA Polymerase-II
. The third subunit of TFIIIB
is a 90-kDa polypeptide called B". Once TFIIIB
has bound, then RNA Pol-III
can bind and initiate transcription in the presence of ribonucleoside triphosphates. Initiation by RNA Polymerase-III
does not require hydrolysis of an ATP Beta-Gamma bond similar to RNA Polymerase-I
. Once TFIIIB
can be removed without affecting initiation by RNA Polymerase-III
serves as an assembly factor for the critical initiation factor, TFIIIB
(Ref.2, 5 & 6).
Transcription and elongation start immediately after assembly of the Initiation Complex. RNA Pol-III
moves along the DNA and matches the DNA nucleotides with a complementary RNA nucleotide to create a new RNA molecule that is patterned after the DNA. The copying of the DNA continues until the RNA Pol-III
reaches a termination signal, which is a specific set of nucleotides that mark the end of the gene to be copied and also signals the disconnecting of the DNA with the newly minted RNA. The initial transcripts produced from tRNA genes are precursor molecules, which are processed into mature tRNAs. Pre-tRNA are processed by cleavage of a 5 leader sequence and by splicing to remove an intron close to the anticodon loop. The enzyme Splicing endonuclease
helps in the process of Splicing. Upon maturation the UU sequence at the 3 end is replaced by CCA. Eukaryotic tRNAs contain a large variety of modified nucleotided for fine tuning of activity, fidelity and stability, which are formed post-transcriptionally. Mature tRNA are then exported to the cytoplasm to take part in the process of protein synthesis (Ref.7 & 8). Cytoplasmic tRNA genes can be grouped into 49 families according to their anticodon features. These genes are found on all chromosomes, except 22 and Y chromosome. High clustering on 6p is observed (140 tRNA genes), as well on 1 chromosome. tRNA is the key to deciphering the code words in mRNA. Each type of amino acid has its own type of tRNA, which binds it and carries it to the growing end of a polypeptide chain if the next code word on mRNA calls for it. The correct tRNA with its attached amino acid is selected at each step because each specific tRNA molecule contains three-base sequences that can base-pair with its complementary code word in the mRNA. The accuracy of charging tRNA with the proper amino acid is crucial because once charged, only the tRNA anticodon determines incorporation, not the attached amino acid (Ref.1, 9 & 10).