RNA : functional role Hamad Yaseen, PhD MLS Department, FAHS Hamad.ali@hsc.edu.kw RNA mrna rrna trna 1
From DNA to Protein -Outline- From DNA to RNA From RNA to Protein From DNA to RNA Transcription: Copying of one strand of DNA into a complementary RNA sequence by the enzyme RNA polymerase. DNA VS. gene? 2
From DNA to RNA Transcription key players: DNA template RNA polymerase mrna From DNA to RNA DNA Template DNA template strand: refers to the sequence of DNA that is copied during the synthesis of mrna. Coding Strand: refers to the strand with a base sequence directly corresponding to the mrna sequence. 3
From DNA to RNA RNA polymerase The enzyme that perform the transcription. It catalyzes the formation of the phosphodiester bonds that link the nucleotides together to form the linear RNA chain. Promoter Transcription unit Transcription 5 3 Start point RNA polymerase 5 3 Unwound DNA 5 3 DNA Initiation RNA Template strand transcript of DNA Elongation Rewound DNA 5 3 3 5 3 5 3 5 The three stages of transcription: Initiation After the RNA polymerase binds to the promoter, the DNA strands unwind, and the polymerase initiates RNA synthesis at the start point of the template strand. Elongation The polymerase moves downstream, unwinding the RNA transcript 5`-3`. In the wake of transcription, the DNA strands re-form the double helix. 5 3 RNA transcript 5 Termination Completed RNA transcript 3 3 5 Termination Eventually, the RNA transcript is released and the polymerase detaches from the DNA. It happens when the polymerase encounters a terminator. 4
The Chain elongation Reaction RNA polymerase catalyzes chain elongation by a mechanism almost identical to the one used by DNA polymerase. The Chain elongation Reaction RNA polymerase catalyses a nucleophilic attack by the 3`-OH group of the growing RNA strand on the alfa phosphorus atom of the dntp. As a result a phosphodister bond forms and pyrophosphate is released. 5
The Chain elongation Reaction a Phosphodister bond : A bond between a two sugar groups and a phosphate group; such bonds form the sugar-phosphate-sugar backbone of DNA and RNA. 6
5' capping, 3' polyadenylation, RNA splicing From DNA to RNA -RNA Processing- Post transcription 5' capping: From DNA to RNA -RNA Processing- Ensures stability of mrna during translation 7
RNA processing 3' polyadenylation The pre-mrna processing at the 3' end of the RNA molecule involves cleavage of its 3' end and then the addition of about 250 adenine residues to form a poly(a) tail. The poly-a tail makes the RNA molecule more stable and prevents its degradation. Additionally, the poly-a tail allows the mature messenger RNA molecule to be exported from the nucleus and translated into a protein by ribosomes in the cytoplasm RNA splicing: From DNA to RNA -RNA Processing- Splicing is a modification of the nascent premessenger RNA (pre-mrna) transcript in which introns are removed and exons are joined 8
Sequences within the RNA Determine Where Splicing Occurs The borders between introns and exons are marked by specific nucleotide sequences within the pre-mrnas. 5 splice site: the exon-intron boundary at the 5 end of the intron 3 splice site: the exon-intron boundary at the 3 end of the intron Branch point site: an A close to the 3 end of the intron, which is followed by a polypyrimidine tract (Py tract). 9
The intron is removed in a Form Called a Lariat as the Flanking Exons are joined Two successive transesterification Step 1: The OH of the conserved A at the branch site attacks the phosphoryl group of the conserved G in the 5 splice site. As a result, the 5 exon is released and the 5 -end of the intron forms a three-way junction structure. Step 2: The OH of the 5 exon attacks the phosphoryl group at the 3 splice site. As a consequence, the 5 and 3 exons are joined and the intron is liberated in the shape of a lariat. Three-way junction 10
RNA splicing: From DNA to RNA -RNA Processing- Alternative splicing : provide alternative transcripts from the same gene which produce multiple proteins. It increase the coding potential of the genome. Humans produce around 150,000 different proteins from their 25,000 30,000 genes. 11
Translation The final stage of biological information flow. The translation of mrna and the polymerization of amino acids into proteins. 12
The Genetic Code Codons: a sequence of three nucleotide residues in mrna or DNA that specifies a particular amino acid according to the genetic code. The Genetic Code Remember synonymous and non- synonymous mutations 13
The Genetic Code Standard Genetic code features: Universality the genetic code is the same in almost all living organisms, from bacteria to mammals Codons are always translated from 5 to 3. Each codon corresponds to only one amino acid. The Genetic Code There are multiple codons for most amino acids Degenerate. For example for Serine there are six codons. Four from glycine, two for lysine. (why this is important?) Different codons that specify the same amino acid are known as synonymous. The only amino acids with single codon are methonine and tryptophan. 14
The Genetic Code Only 61 of the 64 codons specify amino acids. The three remaining codons (UAA, UGA, UAG) are termination codons or stop codons. These codons are not recognized by any trna molecule. Methionine codon AUG also specifies the initiation site for protein synthesis Initiation codon. Three main elements mrna Transfer RNA (trna) Ribosomes Translation 15
trna They are the crucial links between the mrna sequence and the sequence of amino acids in the polypeptide. There is a specific trna for each amino acid. Transfer RNA trna look like threelobed cloverleaf due to base pairing between complementary nucleotides on different regions of each trna molecule causing it to fold 16
trna Structure Amino Acid stem The Amino acid is covalently attached to the 3 of this stem Anticodon Arm Anticodon The anticodon sequence is written in the 3 to 5 direction. 17
trna Anticodons with mrna codons trna and mrna interacts through base pairing between anticodons and codons. Base pairing follows Watson-Crick rules with exceptions!! Wobble position The codon must form watson-crick base pairs with the 3 and middle bases of the anticodon. BUT, other types of base pairing are permitted at 5 position of the anticodon. This postion is called Wobble Position. 18
Wobble position I : Inosine Wobble position 19
Peptide Synthesis Aminoacyl-tRNA Synthetases In Aminoacylation a particular amino acid is covalently attached to the 3 end of each trna molecule. The product of this reaction is called an aminoacyl-trna. 20
In step 1, an O atom of the amino acid a- carboxyl attacks the P atom of the initial phosphate of ATP. In step 2, the 3' OH of the terminal adenosine of trna attacks the amino acid carbonyl C atom. Aminoacyl-tRNA Synthetase - summary: 1. amino acid + ATP aminoacyl-amp + PP i 2. aminoacyl-amp + trna aminoacyl-trna + AMP 21
Ribosomes They are composed of: Ribosomal RNA Protein 22
Ribosome Composition (S = sedimentation coefficient) Ribosome Source Whole Ribosome Small Subunit prokaryotic 70S 30S 16S rrna 21 proteins Eukaryotic 80S 40S 18S rrna 33 proteins Large Subunit 50S 23S & 5S rrnas 31 proteins 60S 28S, 5.8S, &5S rrnas 49 proteins Eukaryotic cytoplasmic ribosomes are larger and more complex than prokaryotic ribosomes. 23
Ribosomes Ribosomes have two Aminoacyl-tRNA Sites: A site P site Initiation of Translation The first codon is usually AUG. There are at least two types of trna that recognize AUG codon. One of them is used exclusively for initiation of translation and is called Initiator trna. 24
Chain elongation Peptidyl Transferase Enzymatic activity of large ribosomal subunit. It catalyzes peptide bond formation. Termination of translation Initiated by termination codons (UGA, UAG, UAA) at A site. These codons are not recognized by trna so protein synthesis stall. Release factors bind to the A site. And the whole complex will get disassociated. 25
mrna trna rrna Types of RNA 26
27