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Macromolecular synthesis
 

Macromolecular synthesis

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  • Good Afternoon, My name is Whitney Barfield and Today we will be discussing Macrmolecular Synthesis: Replication, Transcription and translation. This lecture is normally performed by Dr. Kanaan. However, I will be presenting this lecture today and all of the other lectures related to this topic will be performed by her.
  • Now, We will begin by discussing genetics. Genetics deals with the molecular structure of genes, how they replicate, and how they are inherited from parents to offspring. Genes are segments of deoxyribonucleic acid that encode for functional products called proteins. And proteins facilitate biological reactions.
  • Now genes are coiled very tightly with other proteins called histones into structures called chromosomes, therefore, these chromosomes are said to carry our hereditary information. There the specific collection of an individuals genes is said to be its genotype. Each genotype codes for all of the particular characteristics of an organism. And the proteins that are expressed is referred to as the individuals phenotype. The phenotype describes all of the observable properties of the organism.
  • Now lets talk about DNA. DNA is a type of nucleic acid. The other type being ribonucleic acid or RNA. DNA is a polymer containing several monomers called nuceotides.
  • Nucleotides are composed of three different components—a five carbon sugar called a deoxyribose that has an hydrogen group at its two prime carbon, a phosphate group, and one of four different bases adenine, thymine cytosine and guanine. Now Adenine and guanine are classified as purines because they contain a double ring While pyrimidines cytosine, thymine and Uracil (which replaces thymine in RNA) contain a single ring structure
  • Now the nucleotides join and the sugar and the phosphate group bond making up the DNA backbone—this is called a phosphodiester bond Two separate dna strand align parallel to one another joining at the bases via hydrogen bonding If you looked at it—it would be as if one strand was pointing upward in the 5’ to 3’direction (5’ having a terminal phosphate group and 3’ having a terminal hydroxyl group)
  • Adenine always binds with thymine forming a double hydrogen bon and guanine always binds with cytosine forming a triple hydrogen bond
  • A sugar and a base without a phosphate group is referred to as a nucleoside. The bond between the base and the deoxyribose is referred to as a glycosidic bond.
  • DNA and RNA are similar in that they are carriers of genetic information. However DNA is a much more stable molecule due to the presence of a hydrogen group at the 2’ carbon, while RNA has a much more unstable hydroxyl group at the 2’ carbon. And as I said earlier Uracil replaces thymine in RNA
  • Now in order for this genetic information to be passed during cell division it has to be replicated effectively.
  • Replication has several key steps including pre-priming, priming, elongation and termination.
  • In order for DNA to be replicated, it has to first be separated. To facilitate this separation, proteins referred to as helicases (because they break the helical structure of the DNA) facilitate the separation of the DNA strands. And to ensure that these strands don’t rehybridize, other small proteins called single stranded binding proteins bind to the DNA. This stabilizes the seperation of the two strands.
  • Now as you can see here the helicases have unwound the DNA and the Single stranded binding proteins have bound and stabilized the strands apart from one another forming a replication fork. This makes the bases available for templating reactions.
  • Now, once the replication fork has been formed, an enzyme called a primase is linked directly to the DNA helicase to form a unit called a primosome. The primase can then move along the DNA adding short segments of RNA called primers. Because synthesis always occurs in the 5’ to 3’ direction only one primer is needed for the leading strand (or the DNA strand orientated in the 5’ to 3’ direction) while several primers are needed for the 3’ to 5’ strand.
  • AN enzyme called DNA polymerase three then facilitates the addition of new complementary DNA sequences to the 3’ termini of the primers. The leading strand can be synthesized continuously, while the lagging strand forms short discontinuous DNA fragments called Okazaki fragments.
  • . DNA polymerase 1 then digests the RNA primers and replaces it with DNA, and another enzyme called DNA ligase then fills in the gaps forming a continuous template Now instead of one DNA molecule you have two and that is how replication occurs
  • Now in order for this DNA to be express its encoded protein, it must be transcribed into a single stranded RNA molecule specifically messenger RNA. Messenger RNA is a complementary single stranded copy of the DNA template. Transcription also occurs in the 5’ to 3’ direction.
  • In bacteria transcription begins with the binding of an RNA polymerase consisting of five subunits called a holoenzyme to a binding site called a promoter. The sigma subunit recognizes the Adenine Thymine rich region (or promoter) approximately ten bps upstream the start site of transcription
  • RNA polymerase travels down the template strand and uses base pairing complementary with the DNA template to create an RNA copy. Only one strand of the DNA template will be used for transcription and the RNA complementary copy will have Uracil in the place of thymine.
  • Elongation continues until it reaches an inverted repeat sequence in the DNA template—forming a stem loop structure. Or termination occurs with the help of rho a termination factor which actively unwinds the DNA-RNA hybrid formed during transcription
  • Now after the mRNA transcript is made, it has to undergo translation. Translation is the process of converting an mRNA transcript into an amino acid (polypeptide) sequence that will later fold into its functional protein product. Now in translation there are three types of RNA that are used mRNA which carries the information for the amino acid sequence. rRNA which combine to form the ribosome on which protein synthesis occurs tRNA whose primary role is to capture the appropriate amino acid and bring it to the ribosome and make sure that it is inserted into the right point on the growing amino acid chain
  • As I said earlier polypeptide synthesis occurs on ribosomes. Ribosomes are made up of a large subunit and small subunit. In bacteria the large subunit is 70S and the small subunit is 30S. While in Eukaryotes the large subunit is 60S, while the small suunit is 40S
  • To begin protein synthesis the small subunit of the ribosome binds to the mRNA upstream of the start codon (a codon is three adjacent bases that code for a specific amino acid) (AUG). This is called the Shine Dalgarno sequence.
  • Next, an enzyme called amino-acyl tRNA synthase adds the correct amino acid to its tRNA. When this occurs the tRNA is said to be charged. The tRNA The start codon in translation is AUG and a tRNA

Macromolecular synthesis Macromolecular synthesis Presentation Transcript

  • Macromolecular Synthesis: Replication, Transcription & Translation Yasmine Kanaan, PH. D. 11/06/11
  • Genetics
    • Genetics is the science of heredity
    • It includes the study of
    • what genes are
      • how they carry information,
        • how they are replicated
        • and passed to subsequent generations of cells or passed between organisms,
    11/06/11
    • Chromosomes: are cellular structures that physically carry hereditary information. The chromosomes contain the genes.
    •  
    • Genes: are segments of DNA (except in some viruses, in which they are made of RNA) that code for functional products.
    • Genotype: Is collection of genes
    • Genotype of an organism is its genetic makeup. The information that codes for all the particular characteristics of the organism.
    • Phenotype: Is collection of proteins
    • Refers to actual, expressed properties such as the organism’s ability to perform a particular chemical reaction.
    11/06/11
  • DNA
    • As one of four macromolecules (carbohydrates, lipids, proteins, NA) in the cell, is classified as one type of NA. The other type of NA is RNA.
    • A very large, long molecule is made up of smaller “subunits” called Nucleotides .
    11/06/11
  • Nucleotides
    • Are made up of three components:
      • One of four nitrogenous bases (cyclic compounds made up of C, H, O, N): Adenine (A), Thymine (T), Cytosine C, guanine G.
        • a) Purines : A and G (double-ring structure)
        • b) Pyrimidines : T, C, and U (single-ring structure)
      • A 5-carbon sugar (deoxyribose)
      • A phosphate group
      • Thus,
      • one base + deoxyribose +phosphate=nucleotide
    11/06/11
  • 11/06/11
  • Bases are held together by hydrogen bond
    • A=T
    • G = C
    11/06/11
  • Nucleoside
    • Combination of a purine or pyrimidines plus a pentose sugar.
    • purine+ sugar No phosphate pyrimidine+ sugar group
    • N-glycosidic linkage - refers to the type of bond between the sugar and base
    11/06/11
  • DNA
    • Is a double helical molecule consists of two “strands” that form a “right-handed” spiral
    • The two strands are aligned antiparallel to each other (one seems to pointing downward and other upward)
    • The two strands are held together by hydrogen bonds.
    11/06/11
  • THE GENETIC MATERIAL
    • DNA
      • DNA = deoxyribonucleic acid. The sugars in DNA contain a 2' hydrogen
      • DNA is chemically stable
      • DNA functions as the carrier of genetic information (usually)
      • DNA contains the bases A, G, C, and T
      • RNA
      • RNA = ribonucleic acid. The sugars in RNA contain a 2' hydroxyl group
      • Due to the presence of the 2' hydroxyl group, RNA is less stable than DNA
      • RNA usually functions as the carrier of genetic information
      • RNA contains the bases A, G, C and U
    11/06/11
  • Oligonucleotides
    • Oligonucleotide growth occurs in the 5' to 3' direction via an attack by 3' hydroxyl groups upon the 5' alpha phosphate of nucleotide triphosphates
    • The resulting linkage is a phosphodiester
    •  
    •  
    11/06/11
  • 11/06/11
  • Current Model for DNA Replication
    • The replication of DNA is accomplished by a multienzyme DNA replicase system.
    • There are four stages in the replication process
    • Prepriming (preparation for replication)
    • Priming (synthesis of DNA primers)
    • Elongation (addition of DNA sequences to the 3’-termini of primers)
    • Termination (removal of RNA primers & their replacement w/DNA sequences & the subsequent covalent joining of the DNA fragments).
    11/06/11
  • Cont1. Replication / 1. Prepriming
    • The continuous unwinding of DNA at the replication fork is performed by a number of proteins known as helicases:
    • Helicase II or III binds to the lagging strand
    • The Rep protein also a helicase binds to the leading strands.
    • The subsequent stabilization of the unwound DNA, & prevention of reanealing of the two seperated strands is provided by tetrameric aggregates of SS DNA-binding proteins (SSB).
    • During the progression of replication, the SSB proteins are recycled & bind to new single stranded sites along DNA.
    11/06/11
  • 11/06/11
  • Cont2. Replication / 2. Priming
    • Involves the synthesis of the 5’-3’ RNA primers (length 10 nucleotides)
    • Priming is catalyzed by the enzyme primase
    • Six proteins dnaB, c, I, n, n’, n’’, perform specific functions & together they form an aggregate known as primosome
    11/06/11
  • Cont3. Replication / 3. Elongation
    • Involves the addition of DNA sequences to the 3’-termini of the RNA primers synthesized during priming.
    • The chain elongation process is catalyzed by DNA polymerase III holoenzyme. The complete synthesis of an intact DNA strand is performed with two additional enzymes, DNA polymerase I & DNA Ligase.
    11/06/11
  • Cont4. Replication / 4. Termination
    • During termination degradation of RNA, & elongation of DNA are catalyzed concurrently by two distinct active sites in DNA Polymerase I.
    • DNA Poly. I removes RNA primers (5’-3’ exonuclease activity).
    • DNA poly. I extends the DNA sequence on the 3’-terminus of one of the phosphate group at the 5’-terminus of the other.
    • DNA Ligase catalyzes the formation of a phosphodiester bond bet. two fragments by joining the 3’-OH terminus of one of the phosphate group at the 5’-terminus of the other.
    11/06/11
  • Cont5. Replication / 4. Termination
    • In animal cells and bacteriophage the energy required is provided by ATP hydrolysis. ATP
    11/06/11
  • 11/06/11
  • Transcription
    • The synthesis of a single stranded RNA
    • Depends on a DNA template
    • Occurs in the 5’-3’ direction
    • Produces an antiparallel complementary copy of one of the two strands of the DNA
    • Requires ribonucleoside triphosphates as substrates
    • Is catalyzed by RNA polymerase
    • Advances by pyrophosphate cleavage, & the subsequent hydrolysis of the pyrophosphate to inorganic phosphate.
    11/06/11
  • Cont1 . Transcription
    • The holoenzyme (  2  ’  ) is required for the synthesis of all three RNAs (mRNA, rRNA, tRNA) in E.coli.
    • The sigma factor (  ) recognizes a specific binding site on the DNA strand.
    • The  &  ’subunits function in the binding of the RNA poly complex to the DNA.
    • The binding site is called a promoter
    • The promoter is located about ten bp upstream of the start of the transcription.
    11/06/11
  • Cont2 . Transcription
    • The consensus sequence (A-T-rich seq of six bps) in the promoter serves as the recognition site for RNA poly binding. This seq is known as pribnow box in procaryotes (6bp).
    • In eucaryotes this seq is about 20-30 bps before the start of transcription & is called the hogness or the TATA box.
    11/06/11
  • Cont3 Termination of Transcription
    • Simple termination which is due to stem-&-loop structure in the RNA transcript that was synthesized from an inverted repeat sequence of the gene being transcribed.
    • Termination requiring an auxillary termination factor, a protein called rho.
    • rho depletes the substrate for RNA synthesis by hydrolyzing ribonucleoside triphosphates.
    11/06/11
  • 11/06/11
  • 11/06/11
  • Translation
    • Phases in polypeptide synthesis
    • Amino acid activation
    • Initiation of polypeptide synthesis
    • Elongation of a polypeptide chain
    • Termination of polypeptide synthesis
    11/06/11
  • Cont1. Translation
    • Polypeptide synthesis occurs on ribosomes (70 S)
    • The 70 S ribosome of E.coli is composed of a 30 S (small) subunit & a 50 S (large) subunit.
    • The 30 S subunit is composed of 16 S RNA and 21 proteins.
    • 50 S subunit contains two RNAs (23 S & 5S) and 32 proteins.
    • Polypeptide synthesis occurs on the head
    • & platform regions of the
    • 30 S subunit & the upper
    • half of the 50 S subunit.
    11/06/11
  • Cont2 . Translation
    • The 30 S subunit is the site of attachment for both mRNAs and tRNAs.
    • The peptidyl transferase site in the central region of the 50 S subunit.
    11/06/11
  • Cont3 . Translation 1. Amino acid activation
    • In the Ist step an aminoacyladenylate is synthesized by joining the carboxyl group of an amino acid w/ the alpha phosphate of ATP.
    • The Rxn is catalyzed by aminoacyl-tRNA synthetase & the required energy is furnished by the cleavage of pyrophosphate of ATP, & the hydrolysis of the pyrophosphate to inorganic phosphate.
    11/06/11
  • Cont4 . Translation 1. Amino acid activation
    • In the 2 nd step the aa of the adenylate derivative is transferred to a hydroxyl group of the 3’-terminal adenyl nuceotide of a tRNA.
    • The Rxn is catalyzed by aminoacyl-tRNA synthetase.
    • AA+ ATP+ tRNA +H 2 0==Aminoacyl-tRNA+ AMP+ 2Pi
    • AA +
    11/06/11 enzyme AA ATP
  • Cont5 . Translation 2. Initiation of Polypeptide Synthesis
    • An initiation complex consisting of a 30 S subunit, an initiation tRNA carrying N-formylmethionine, & mRNA is formed in the initial step.
    • 3 protein initiation factors (IF1, IF2 & IF3), & GTP are involved in this step. Formylated methionine is the aa that initiates polypeptide synthesis in E.coli .
    11/06/11
  • Cont6 . Translation 2. Initiation of Polypeptide Synthesis
    • In the 2 nd step of initiation, the 50 S ribosomal subunit combines w/ the 30 S initiation complex to form the 70 S initiation complex.
    • During this step GTP is hydrolyzed to GDP and Pi, & the three initiation factors are released from the complex .
    11/06/11
  • Cont7 . Translation Elongation of a Polypeptide Chain
    • Elongation is mRNA directed & proceeds from the N-terminus to the C-terminus of the polypeptide being synthesized.
    • The ist step of elongation involves the binding of an aminoacyl-tRNA/EFTu-GTP complex to the recognition (R) site of the small (30 S) subunit of a functional ribosome. EFTu is a temperature unstable elongation factor and promotes the hydrolysis of GTP to GDP and Pi.
    11/06/11
  • Cont8 . Translation Elongation of a Polypeptide Chain
    • The aminoacyl-tRNA is transferred to a second site in the 30 S subunit known as aminoacyl (A) site.
    • A peptide bond is formed between the two aa residues attached to the tRNAs occupying the P & A sites on the 70 S initiation complex.
    11/06/11
  • 11/06/11
  • 11/06/11
  • Cont9 . Translation Elongation of a Polypeptide Chain
    • Peptidyl transferase catalyzes the peptide bond formation & transfer of the N-formylmethionyl residue at the P site to the NH 3 group of the aa residue attached to the aminoacyl tRNA at the A site, producing a dipeptidyl-tRNA.
    • The tRNA w/ out aa at the P site is released, & the peptidyl-tRNA is relocated to the P site by another elongation factor called EFG or transloase.
    11/06/11
  • Cont10 . Translation Termination of a polypeptide bond
    • The termination of a polypeptide synthesis is signaled by one of the 3 termination codons (UAA, UAG, or UGA) at the end of an mRNA.
    • The 3-base sequence of a termination codon is recognized by one of the two release factors, RF1 (which recognizes UAA or UAG) of RF2 (which recognizes UAA or UGA).
    • Both release factors function in combination w/ GTP, which is hydrolyzed to GDP an Pi during termination.
    11/06/11
  • 11/06/11 Genetic code: determines how a nucleotide sequence is converted into the sequence of protein.
  • 11/06/11