Lecture 9 Nucleic Acids II PDF

Nucleic acids II LECTURE 9 DR. DINA NADA  Remember the Central Dogma The genetic master plan (genetic information) of an organism is contained in the DNA mRNA is the working copy of the DNA through which the master plan is expressed The copying process, during which a DNA strand serves as a...

Nucleic acids II LECTURE 9 DR. DINA NADA  Remember the Central Dogma The genetic master plan (genetic information) of an organism is contained in the DNA mRNA is the working copy of the DNA through which the master plan is expressed The copying process, during which a DNA strand serves as a template for the synthesis of mRNA is called Transcription mRNAs are then translated into proteins in a process called Translation (protein synthesis) RNA (Ribonucleic acids) All RNAs are single stranded polynucleotides Types of RNA: ▪ Messenger RNA= mRNA ▪ Transfer RNA= tRNA Are involved in the translation process ▪ Ribosomal RNA= rRNA ▪ Small RNA molecules Molecules that perform structural, catalytic and regulatory functions Structure of RNA Building blocks of RNA ▪ RNA is a polynucleotide compound ▪ Nucleotide = Nitrogenous base (A,G,C,U) + sugar (ribose)+ monophosphate ▪ These nucleotides are joined together by phosphodiester bonds ▪ They exist as single strands that are capable of folding into complex structures Messenger RNA (mRNA) mRNA is the working copy of the DNA through which the genetic information is expressed (complementary to the DNA strand from which it has been transcribed) Carries genetic information from DNA to cytosol for protein synthesis Make up about 5% of the total RNA in the cell Each 3 successive nucleotides is a codon for a specific amino acid  Transfer RNA (tRNA) It is an adaptor molecule that carries special amino acid Make up about 15% of the total RNA in the cell It has 3 loops and 2 free ends There is an extensive interchain base pairing The anticodon region complementary to codons on mRNA Ribosomal RNA (rRNA) They are found in association with several proteins as components of the ribosomes (the complex structures that serve as the sites for protein synthesis) Make up about 80% of the total RNA in the cell There are distinct size species of rRNA: ▪ Three in prokaryotes: 5S, 23S and 16S ▪ Four in eukaryotes: 5S, 5.8S, 28S and 18S ▪ S is the Svedberg unit for sedimentation rate (determined by size and shape of the particle) One type One type RNA transcription It means mRNA synthesis RNA transcription requires: 1) DNA template composed of: ▪ Promoter region ▪ Transcription unit ▪ Termination unit 2) Ribonucleotides phosphates A,G,C,U 3) RNA polymerase  Promoter region (Read only) Promoters have sequences that are recognized by the RNA polymerase enzyme Prokaryotic promoters has: ▪ Pribnow box: Stretch of six nucleotides (5'-TATAAT-3') centered about ten nucleotides to the left of the transcription start site A (-10) ▪ A second nucleotide sequence (5’-TTGACA-3’) is centered about 35 bases to the left of the transcription start site (-35) ▪ 19 base pairs in between the 2 sequences Eukaryotic promoters has: ▪ TATA box: Stretch of nucleotides identical to pribnow, 25 nucleotides to the left of the transcription start site ▪ CAAT box 70-80 bases to the left of the transcription start site ▪ 40 base pairs in between the 2 sequences Prokaryotic RNA polymerase RNA polymerase enzyme is responsible for the synthesis of RNA molecules In bacteria, one species of RNA polymerase synthesizes all types of RNA molecules except RNA primers (needed for DNA replication) Eukaryotic RNA polymerases There are three species of RNA polymerase in eukaryotic cells: RNA polymerase I: synthesizes the large rRNAs in the nucleolus RNA polymerase II: which synthesizes mRNA and recognizes the promoter region RNA polymerase III: which produces the small RNAs including the tRNA and the small 5s ribosomal RNA Steps of transcription Initiation Elongation Termination Initiation 1- At first, RNA polymerase binds with the promoter area 2- Binding of RNA polymerase to DNA template leads to local separation (unwinding) of the DNA double helix into sense and antisense strands  Elongation 1- At the antisense strand, formation of RNA molecule begins at the 5` end by RNA polymerase enzyme 2- Then elongation of the RNA molecule occurs from 5` to 3` end, antiparallel to its template 3- The nucleotide building blocks are 5` ribonucleoside triphosphates (ATP, GTP, CTP and UTP). Pyrophosphate (PPi) is released when each new nucleotide is added to the growing chain e.g. GTP = GMP +PPi 4- RNA polymerase forms a phosphodiester bond between the 3` OH of one ribose sugar and 5` OH of the next ribose 5- The process of elongation of RNA chain continues until a termination region reached  Termination 1- Termination region on DNA template can be recognized by: a- RNA polymerase enzyme itself (rho independent termination) b- Rho factor, which may be required for the release of both RNA strand and RNA polymerase (rho dependent termination) 2- Termination results from: a- Binding of rho factor to polymerase enzyme: When RNA polymerase enzyme reaches the termination site, rho factor binds with it causing termination b- Slowing down of RNA polymerase at the termination site: at palindromes Palindromes Termination site on DNA is characterized by the presence of palindromes 5` 3` Palindrome is a region of a double stranded DNA in which each of the two strands has the same 3` 5` sequence when read in the same direction e.g. in the 5` to 3` direction The RNA transcript of the DNA palindrome can form a stable hairpin structure, which is a self complementary structure. This hairpin structure causes slowing down of RNA polymerase at the termination site  mRNA Post transcriptional modifications Pre-m RNA is modified into mature mRNA in the nucleus by: 1- 5’ Capping a) The addition of 7-methylguanosine triphosphate to the 5’- terminal end of the m-RNA b) It facilitates the initiation of translation c) Protects the 5` end of mRNA from 5` to 3’ exonucleases 2- Addition of A poly-A tail a) A chain of 40-250 adenine nucleotides is attached to the 3’ end UTR: Untranslated region b) This protects the 3` end of mRNA from 3`to 5` exonuclease 3- Splicing Spliceosomes are structures responsible for removal of introns from the pre-mRNA, and ligation of both ends of exons to form mature mRNA Genetic code The genetic information = the sequence of bases of the DNA These bases are organized into 3 letter code words called codons Each codon codes for a specific amino acid The collection of these codons makes up the genetic code (dictionary that gives the relationship between the sequence of bases in DNA (or in transcribed mRNA) and a sequence of amino acids in a protein RNA copy (transcript) of a specific sequence of DNA (gene) can be translated into a sequence of amino acids in the cytoplasm to form a polypeptide or protein Genetic code Codons can be defined as: The sequence of 3 nucleotide bases on mRNA which determines the type and position of the amino acid that well enter in the structure of protein molecule. They are read from 5’ to 3’ There are 64 codons 3 termination codons (nonsense) UAG, UGA, UAA codes for no aa and terminate protein synthesis AUG is the start codon and it codes for Methionine 61 codons code for 20 aa Read only Characteristics of the genetic code 1) Specificity: specific codon always codes for only a single amino acid 2) Degeneracy: multiple codons must code for the same amino acid 3) Nonoverlapping: the code is read from a fixed starting point as a continuous sequence of bases, taken 3 bases at a time. If one deleted, change reading frame 4) Universal: for all species of plants and animals with the exception for mitochondria: AUA codes for Trp instead of Ile and UGA codes for Trp instead of acting as stop codon Codon-anticodon recognition Recognition of a particular codon on the mRNA by the anticodon sequence of the tRNA The binding of tRNA anticodon to the mRNA codon follows complementary, antiparallel When writing the sequence of both codons and anticodons, the nucleotide sequence must always be listed in the 5` to 3` order Translation (protein synthesis) Components required for protein synthesis: 1- Amino acids 2- Transfer RNA (tRNA) 3- Messenger RNA (mRNA) 4- Aminoacyl-tRNA synthetase enzyme: ▪ An enzyme that attaches the appropriate amino acid onto its tRNA 5- Protein factors: several protein factors including initiation, elongation and termination factors 6- Ribosomes  Ribosomes Ribosomes are large complexes of proteins and rRNAs They serve as factories responsible for protein synthesis There are two subunits of eukaryotic ribosomes (60S and 40S) The complete ribosome has two binding sites for tRNA molecules. The A site and the P site, each of which extends over both subunits, and an E site for the release of uncharged tRNA Together they cover two neighboring codons on the mRNA  Steps of protein synthesis Protein synthesis translates the 3 letters alphabet (codons) on mRNA into amino acids that constitutes the protein mRNA translated from 5’-3’ Protein synthesized from NH2 terminal to COOH terminal The main steps are: A) Initiation B) Elongation C) Termination A) Initiation Involves assembly of the components before peptide bond formation: ▪ Ribosomes ▪ mRNA to be translated ▪ aminoacyl-tRNA specified by 1st codon on mRNA (AUG for methionine) ▪ Initiation factors The initiating AUG is recognized by a special initiator tRNA The initiator tRNA-aa enters in the P-site Initiator tRNA carries methionine Now the P- site is occupied by met-tRNA, and A-site is free and ready to receive the subsequent aminoacyl tRNA B) Elongation Involves addition of aa to the COOH end of the growing polypeptide chain The aminoacyl-tRNA whose codon appear next on mRNA template will be delivered to A- site The carboxyl group (-COOH) of the aminoacyl-tRNA in P site is transferred to and bind with the amino (NH2) group of the new aminoacyl tRNA in the A site. This reaction is catalyzed by peptidyl transferase enzyme present in 60S subunit Elongation Translocation: After peptide bond is formed, the ribosomes move 3 bases towards the 3’end of mRNA As a result of movement of ribosomes, the following events occur: 1) Transfer of the newly formed peptidyl tRNA from A site to occupy P-site 2) Release of the uncharged tRNA from E- site of ribosome 3) A site becomes free. Thus it can be occupied by another new aminoacyl tRNA according to the codon-anticodon recognition Then the process will be repeated C) Termination Termination is the final step which occurs after multiple cycles of elongation and formation of the protein Termination occurs when ribosome moves to bring one of the three termination (nonsense) codons into A-site. These codons are UAA, UAG and UGA Releasing factors (eRF) 1, 2 and 3 which are present in A site can recognize all the three termination codons Releasing factors promote the hydrolysis of the bond between peptide and tRNA occupying the P site This hydrolysis leads to: 1) Release of both peptide and tRNA 2) Dissociation of 80S ribosomes into its 40S and 60S subunits  Polysomes Because of the big length of the nucleotide sequence of most mRNAs , more than one ribosome can translate the same mRNA at the same time. Such a complex of one mRNA and a number of ribosomes is called a polyribosome or polysome  Inhibition of protein synthesis Antibiotics: Ribosomes in bacteria are smaller than that of eukaryotes (70S rather than 80S). Bacteria also have simpler components of RNA and protein molecules. This allows many antibiotics react specifically with bacterial ribosomes and thus inhibit protein synthesis. This results in bacterial death without harmful effect on eukaryotic cell Examples of these antibiotics are: 1- Streptomycin: It binds to the ribosome, distorting its structure. It causes dissociation of mRNA from the ribosomes 2- Tetracycline: They interact with small ribosomal subunits and prevent aminoacyl tRNA anticodons from recognizing their corresponding codons 3- Chloramphenicol: It inhibits peptidyl transferase enzyme 4- Puromycin: Its structure resembles the structure of aminoacyl tRNA. It becomes incorporated into the growing peptide chain, thus causing inhibition of further elongation Diphtheria toxins: It is an exotoxin produced by a bacteria called corynebacterium diphtheria, these toxins inactivate the eukaryotic elongation factor-2 (eEF-2) thus preventing translocations