Chapter 4 DNA, RNA, and the flow of genetic information

Chapter 4 DNA, RNA, and the flow of genetic information

The Story of “Molecular Biology”

Watson and Crick (1953)

Structure of DNA & DNA Replication
DNA Structure Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Watson, J.D., and Crick, F.H. Nature (April 1953) 171(4356): DNA Replication Genetical implications of the structure of deoxyribonucleic acid. Nature (May 1953) 171(4361): Nobel Prize in Medicine (1962)

Evidence for the Structure of DNA
Evidence Available to Watson and Crick: Nucleotides & Nucleotide Pairing (Pyrimidine + Purine) Chargaff’s Rule X-ray Diffraction Analysis (Rosalind Franklin and Maurice Wilkins) Rosalind Franklin

Molar Properties of Bases
Organism Adenine Thymine Guanine Cytosine AT/GC Ratio E. coli 26.0 23.9 24.9 25.2 1.00 D. pneumoniae 29.8 31.6 20.5 18.0 1.59 M. tuberculosis 15.1 14.6 34.9 35.4 0.42 Yeast 31.3 32.9 18.7 17.1 1.79 Sea Urchin 32.8 32.1 17.7 18.4 1.85 Herring 27.8 27.5 22.2 22.6 1.23 Rat 28.6 28.4 21.4 21.5 1.33 Human 30.9 29.4 19.9 19.8 1.52

Chargaff’s Rule Another set of clues available to Watson and Crick came from the work done by Erwin Chargaff, who studied the nucleotide composition of DNAs from different organisms. Chargaff established certain “rules” about the amounts of each component of DNA: The total amount of pyrimidine nucleotides (T + C) always equals the total amount of purine nucleotides (A + G). The amount of T always equals the amount of A, and the amount of C always equals the amount of G. But the amount of A + T is not necessarily equal to the amount of G + C. This ratio varies among different organisms.

Wilkins and Franklin (1952)

DNA Replication SEMI-CONSERVATIVE REPLICATION: 2 new DNA helices are produced Each helix has 2 DNA strands One strand is from the parental DNA (PURPLE) The other strand is newly synthesized (BLUE)

Nucleic Acids

Nucleic Acids Nucleic acids store, transmit, and use genetic information. DNA = deoxyribonucleic acid & RNA = ribonucleic acid The monomers of nucleotides consist of a pentose sugar, a phosphate group, and a nitrogen-containing base.

Nucleic Acids RNA has “ribose” & DNA has “deoxyribose”
The “backbone” of DNA and RNA is a chain of sugars and phosphate groups, bonded by PHOSPHODIESTER LINKAGE. The phosphate groups link carbon 3′ in one sugar to carbon 5′ in another sugar. The two strands of DNA run in opposite directions (antiparallel).

Deoxyribonucleosides

Ribonucleosides

Nucleotide Structure Ring structures are found in both the base and the sugar Base rings are numbered as usual Sugar ring numbers are given the “prime” designation Phosphodiester Bond: covalent bond between the sugar and phosphoryl group -N-glycosidic Bond: covalent bond between the 1'-carbon of the sugar and a nitrogen atom of the base Phosphodiester Bond

Adenosine Triphosphate (ATP)
Adenosine Diphosphate Adenosine Monophosphate Adenosine “Nucleoside” vs. “Nucleotide”

Nucleotide Nomenclature

DNA Double Helix

DNA Double Helix BASE PAIRING: a non-covalent attraction aiding in maintaining the double helix structure is hydrogen bonding between base pairs Adenine forms 2 hydrogen bonds with Thymine: A = T Cytosine forms 3 hydrogen bonds with Guanine: G ≡ C The two DNA strands are COMPLEMENTARY strands - the sequence of bases on one strand automatically determine the sequence of bases on the other strand The chains run ANTIPARALLEL and this is the only structure that allows base pairs to form the Hydrogen Bonds that hold them together

Antiparallel DNA Strands
The DNA Double Helix Single RNA Strand Antiparallel DNA Strands

The Structure of DNA DNA:
3’,5’-phosphodiester bridges link nucleotides together to form polynucleotide chains. The 5’-ends of the chains are at the top; the 3’- ends are at the bottom.

The Structure of RNA RNA:
3',5'-phosphodiester bridges link nucleotides together to form polynucleotide chains. The 5'-ends of the chains are at the top; the 3'- ends are at the bottom.

The DNA Double Helix A, B, Z form of DNA (table 4.2)
Three-dimensional structure of B-DNA. This model shows the orientation of the base pairs and the sugar–phosphate backbone and the relative sizes of the pyrimidine and purine bases. The sugar–phosphate backbone winds around the outside of the helix, and the bases occupy the interior. Stacking of the base pairs creates two grooves of unequal width, the major and the minor grooves. The diameter of the helix is 2.37 nm, and the distance between base pairs is 0.33 nm. The distance to complete one turn is 3.40 nm. (For clarity, a slight space has been left between the stacked base pairs, and the interactions between complementary bases are shown schematically.) A, B, Z form of DNA (table 4.2)

The Chromosome

The Eukaryotic Chromosome
Eukaryotes (number and size of chromosomes vary) True nucleus enclosed by a nuclear membrane Nucleosome which consists of a strand of DNA wrapped around a disk of histone proteins (DNA appears like “beads-on-a-string”) String of beads then coils into a larger structure called the 30 nm fiber With additional proteins next coiled in to a 200 nm fiber Beads-on-a-String

The Histones

Amino acid sequence of the amino-terminus of Histone H4.
Acetylation and deacetylation of lysine side chains. Histone acetylation is linked to transcriptional activation and associated with euchromatin. N-Acetyl Lysine

The Histones

The Nucleosome Each nucleosome is composed of a core particle plus histone H1 and linker DNA. The nucleosome core particle is composed of a histone octamer and about 146 bp of DNA. Linker DNA consists of about 54 bp. Histone H1 binds to the core particle and to linker DNA.

Histone-Depleted Chromosome

DNA Replication

The Central Dogma of Molecular Biology
Replication The two functions of DNA comprise the Central Dogma of Molecular Biology: DNA can reproduce itself (replication) DNA can copy its information into RNA (transcription) RNA can specify a sequence of amino acids in a polypeptide (translation)

DNA Replication DNA must be replicated before a cell divides, so that each daughter cell inherits a copy of each gene Cell missing a critical gene will die Essential that the process of DNA replication produces an absolutely accurate copy of the original genetic information Mistakes made in critical genes can result in lethal mutations Structure of the DNA molecule suggests the mechanism for accurate replication An enzyme could “read” the nitrogenous bases on one strand of a DNA molecule adding complementary bases to a newly synthesized strand SEMICONSERVATIVE Replication: one DNA strand is the original (PARENT) strand and the other is newly synthesized (DAUGHTER) strand Compare to CONSERVATIVE and DISPERSIVE replication.

DNA Replication SEMICONSERVATIVE REPLICATION: 2 new DNA helices are produced Each helix has 2 DNA strands One strand is from the parental DNA (PURPLE) The other strand is newly synthesized (BLUE)

Meselson-Stahl Experiment
14N and 15N are stable (non-radioactive) isotopes, although 14N is far more abundant in nature (99.6%). CeCl Gradient – Buoyancy 20 min./division x 1 hr./60 min. = 3 divisions/hour; 15 generations = 5 hours of E.coli cell division 14N and 15N are stable (non-radioactive) isotopes, but 14N is far more abundant in nature (99.6%).

Centrifugation of DNA in a cesium chloride (CsCl) gradient……a DENSITY gradient. Cultures grown for many generations in 15N and 14N media provide control positions for heavy (15N) and light (14N) DNA bands. When the cells grown in 15N are transferred to a 14N medium, the first generation produces an intermediate DNA band and the second generation produces two bands: one intermediate and one light. Hand-draw “semi-conservative” mode of DNA replication and experimental proof with expected CsCl banding patterns.

What method of DNA replication? The results of Meselsohn and Stahl’s experiments prove that only the semi-conservative model of DNA replication is possible. Semi-Conservative Replication: A single intermediate (14N/15N hybrid DNA) band is produced in the first generation. One intermediate (14N/15N hybrid DNA) and one light (14N/14N DNA) band in the second generation. Hand-draw “conservative” and “dispersive” mode of replication and experimental proof with expected CsCl banding patterns. Contrast with “semi-conservative” replication from previous slide.

The Replisome The Replication Machinery
Topoisomerase (phosphodiester bonds) Single-Stranded DNA-Binding Protein Helicase (hydrogen bonds) RNA Primase 5. RNA Primer 6. Okazaki Fragment 7. DNA Polymerase III 8. DNA Polymerase I 9. DNA Ligase Topoisomerase: an enzyme that can cut and re-form “polynucleotide backbones” in DNA to allow it to assume a more relaxed configuration. Helicase: an enzyme that breaks “hydrogen bonds” in DNA and unwinds it during movement of the replication fork.

Supercoiled DNA & Topoisomerases
The DNA molecule on the left is a relaxed closed circle; breaking the DNA helix and unwinding it by two turns before re-forming the circle produces two SUPERCOILS. Positively supercoiled (extra-twisted) regions accumulate ahead of the replication fork as the parental strands separate for replication. This “supercoiling” can be created or relaxed by TOPOISOMERASES.

Leading Strand vs. Lagging Strand
Events: Helicase: “opening” of replication fork ssDNA-binding protein Primase: RNA Primer Leading Strand : one RNA primer Lagging Strand: Okazaki fragments DNA Pol III: extension of primer DNA Pol I: fill RNA primer region DNA Ligase: joins 3’ and 5’ ends Left: Unwinding of DNA by “Helicase”, prevention of DNA re-annealing by “Single-Stranded DNA Binding Proteins”, RNA primers by “Primase” for leading and lagging strand synthesis. Right: Lagging Strand synthesis by extension of multiple “Okazaki Fragments” (or RNA primers), removal of RNA by “DNA Pol I”, joining of “Okazaki Fragments” by “DNA Ligase” LEADING STRAND SYNTHESIS: a single RNA primer is produced at the replication origin. DNA polymerase III catalyzes the addition of nucleotides in the 5'- 3' direction.

The Replication Fork The two newly synthesized strands have opposite polarity. On the leading strand, 5'  3' synthesis moves in the same direction as the replication fork; on the lagging strand, 5'  3' synthesis moves in the opposite direction.

Lagging Strand Synthesis
A short piece of RNA (BROWN) serves as a primer for the synthesis of each Okazaki fragment. The length of the Okazaki fragment is determined by the distance between successive RNA primers.

Many RNA primers are needed as the replication fork moves DNA polymerase III catalyzes the elongation of the new strand (5'  3' direction) Final steps: The removal of the RNA primers by DNA Polymerase I Filling in the gaps by DNA Polymerase I Sealing the fragments into an intact strand of DNA by DNA Ligase

Summary of DNA Replication
The two DNA strands being replicated are ANTIPARALLEL to one another DNA polymerase III can only catalyze in the 5'  3' direction However, the replication fork moves in one direction with both strands replicated simultaneously (LEADING & LAGGING Strands) Small RNA primers are needed for a starting point of DNA replication RESULT: there are different mechanisms for replication of the two strands a. CONTINUOUS Replication: the leading strand is replicated in one long segment b. DISCONTINUOUS Replication: the lagging strand is replicated in many small segments

Transcription

RNA Transcription in E. coli
Transcription of E. coli ribosomal RNA genes. The genes are being transcribed from left to right. The nascent rRNA product associates with proteins and is processed by nucleolytic cleavage before transcription is complete.

Classes of RNA Molecules
1. Messenger RNA (mRNA) mRNA directs the amino acid sequence of proteins A complimentary copy of a gene It has the codon for an amino acid in a protein 2. Ribosomal RNA (rRNA) Structural and functional component of the ribosome Forms ribosomes by reacting with proteins 3 types in prokaryotes 4 types in eukaryotes 3. Transfer RNA (tRNA) Transfers amino acids to the site of protein synthesis

The RNA Content of an E.coli Cell

Transcription Transcription is catalyzed by RNA Polymerase
Produces a copy of only one DNA strand Stages of transcription: 1. Initiation: RNA polymerase binds to the promoter region at the beginning of the gene 2. Chain Elongation: formation of a 3‘  5' phosphodiester bond, generating a complementary copy 3. Termination: the final step of transcription, the RNA polymerase releases the newly formed RNA molecule

Stages of Transcription
Initiation Elongation Termination

Gene Orientation Orientation of a gene. The sequence of a hypothetical gene and the RNA transcribed from it are shown. By convention, the gene is said to be transcribed from the 5' end to the 3' end, but the template strand of DNA is copied from the 3' end to the 5' end. Growth of the ribonucleotide chain proceeds 5‘  3'.

Post-transcriptional Processing of mRNA
Prokaryotes release a mature mRNA at the end of termination for translation Eukaryote mRNA is a primary transcript which still must be processed in post-transcriptional modification, a three step process: a. A 5' cap structure is added This structure is required for efficient translation of the final mRNA b. A 3' poly(A) tail (100 to 200 units) is added by poly(A) polymerase Poly(A) tail protects the 3' end of the mRNA from enzymatic digestion Prolongs the life of the mRNA c. Splicing

Modification to the 5' end of the mRNA with 7-Methyl-Guanosine (m7G)
5'-Methylated Cap Modification to the 5' end of the mRNA with 7-Methyl-Guanosine (m7G)

Polyadenylation

RNA Splicing SPLICING: removal of portions of the mRNA transcript that are not protein coding 1. Bacterial chromosomes are “continuous” as all DNA sequences from the chromosome is found in the mRNA 2. Eukaryotic chromosomes are “discontinuous” There are extra DNA sequences within the genes that do not encode any amino acid sequence called INTRONS (intervening sequences) Presence of introns makes direct translation to synthesize proteins impossible 3. The introns are cut out and the EXONS (coding sequences) are spliced together

mRNA Splicing

mRNA Splicing & Post-Translational Modification
Triose phosphate isomerase gene from maize and the encoded enzyme. Diagram of the gene showing 9 exons and 8 introns. Some exons contain both translated and untranslated sequences.

Protein Translation

Classes of RNA Molecules
1. Messenger RNA (mRNA) mRNA directs the amino acid sequence of proteins A complimentary copy of a gene It has the codon for an amino acid in a protein 2. Ribosomal RNA (rRNA) Structural and functional component of the ribosome Forms ribosomes by reacting with proteins 3 types in prokaryotes 4 types in eukaryotes 3. Transfer RNA (tRNA) Transfers amino acids to the site of protein synthesis

Protein Synthesis 1. Protein synthesis is called TRANSLATION and is carried out on RIBOSOMES rRNA Proteins 2. Protein synthesis occurs in multiple places on one mRNA at a time mRNA plus the multiple ribosomes are called a POLYSOME tRNA (with ANTICODON) recognizes the appropriate CODON on the mRNA 4. AMINOACYL tRNA SYNTHETASE An enzyme that catalyzes the “esterification” of a specific amino acid or its to its partner tRNA to form an aminoacyl-tRNA Sometimes called “charging” the tRNA with the amino acid. Once the tRNA is charged, a ribosome can transfer the amino acid from the tRNA onto a growing peptide, according to the genetic code.

The Genetic Code The message on DNA that has been translated to mRNA:
1. Degenerate: more than one codon can code for the same amino acid 2. Specific: each codon specifies a particular amino acid 3. Non-Overlapping: None of the bases are shared between consecutive codons No non-coding bases appear in the base sequence 4. Universal: all organisms use the same code

The Genetic Code Degenerate & Specific: 64 codons encode 61 amino acids & 3 “stop” signals Multiple codes for an amino acid tend to have two bases in common Codons are written in a 5'  3' sequence

Non-overlapping Code a. In an overlapping code, each letter is part of three different three-letter. b. In a non-overlapping code, each letter is part of only one three-letter word.

The Reading Frame One mRNA contains three different reading frames. The same string of letters read in three different reading frames will be translated into three different “messages” or protein sequences. Thus, translation of the correct message requires selecting the correct reading frame.

Schematic of the Translation Process

Ribosomes Ribosome: the “workbench” of the cell; holds mRNA and charged tRNAs in the correct positions to allow assembly of polypeptide chain. Composed of rRNA + Proteins Two subunits (held together by ionic bonds and hydrophobic forces) Small ribosomal subunit: 1 rRNA + 33 proteins Large ribosomal subunit: 3 rRNA + 49 proteins Polysome: many ribosomes on one mRNA when many copies of the protein are being made simultaneously

Ribosome Structure Large subunit has three tRNA binding sites:
1. A (aminoacyl) site binds with anticodon of charged tRNA 2. P (polypeptide) site is where tRNA adds its amino acid to the growing chain 3. E (exit) site is where tRNA sits before being released from the ribosome.

Ribosome Structure

Eukaryotic vs. Prokaryotic Ribosomes
The prokaryotic and eukaryotic ribosomes consist of two subunits, each of which contains ribosomal RNA and proteins. The large subunit of the prokaryotic ribosome contains two molecules of rRNA: 5S and 23S. The large subunit of almost all eukaryotic ribosomes contains three molecules of rRNA: 5S, 5.8S, and 28S. The sequence of the eukaryotic 5.8S rRNA is similar to the sequence of the 5' end of the prokaryotic 23S rRNA.

Ribosome Structure Sites for tRNA binding in prokaryotic ribosomes. During protein synthesis, the P (“polypeptide”) site is occupied by the tRNA molecule attached to the growing polypeptide chain, and the A site holds an “aminoacyl-tRNA”. The growing polypeptide chain passes through the tunnel of the large subunit.

tRNA There is at least one tRNA for each amino acid to be incorporated into a protein tRNA is single-stranded molecule with about 80 nucleotides The overall structure is called a “cloverleaf” a. Intrachain hydrogen bonding (A=U and G=C) occurs to give: Regions called stems with an -helix A type of L-shaped tertiary structure The 3'-OH group of the terminal nucleotide can covalently bind the amino acid c. 3 nucleotides at the base of the cloverleaf serve as the “anticodon”, which forms hydrogen bonds to a “codon” on mRNA

tRNA (A) The cloverleaf structure, a convention used to show the complementary base-pairing (red lines). The ANTICODON is the sequence of three nucleotides that base-pairs with a CODON in mRNA. The amino acid matching the codon/anticodon pair is attached at the 3′ end of the tRNA. tRNAs contain some unusual bases, which are produced by chemical modification after the tRNA has been synthesized. For example, the bases denoted Ψ (pseudouridine) and D (dihydrouridine) are derived from uracil. (B and C) Views of the actual L-shaped molecule, based on X-ray diffraction analysis. All tRNAs have very similar structures. (D) The linear nucleotide sequence of the molecule, color-coded to match A, B, and C.

Tertiary Structure of tRNA
The cloverleaf-shaped molecule folds into this three dimensional shape. The tertiary structure of tRNA results from base pairing between the TC loop and the D loop, and two stacking interactions that (left) align the TC arm with the acceptor arm, and (right) align the D arm with the anticodon arm.

The Genetic Code The CODONS are always written with the 5′-terminal nucleotide to the left. Note that most amino acids are represented by more than one codon, and that there are some regularities in the set of codons that specifies each amino acid. Codons for the same amino acid tend to contain the same nucleotides at the first and second positions, and vary at the third position. UAA, UAG and UGA codons do not specify any amino acid but act as termination sites (STOP CODONS), signaling the end of the protein-coding sequence. AUG acts both as a START CODON, signaling the start of a protein-coding message, and also as the codon that specifies METHIONINE.

Base Pairing at the Wobble Position

Base Pairing at the Wobble Position
The tRNAAla molecule with the anticodon “IGC” can bind to any one of three codons specifying alanine (GCU, GCC, or GCA) because I can pair with U, C, or A. Note that the RNA strand containing the codon and the strand containing the anticodon are antiparallel.

Transcription and Translation

Protein Translation 1. Initiation
Initiation factors (proteins), mRNA, initiator tRNA, and small and large ribosomes come together Ribosome has two sites to bind tRNA a. P site binds to the growing peptide b. A site binds the aminoacyl tRNA 2. Chain Elongation: a three step process An aminoacyl tRNA binds to A site Peptide bond formation occurs catalyzed by peptidyl transferase Translocation (movement) of ribosome down the mRNA chain next to codon Shifts the new peptidyl tRNA from the A site to the P site Chain elongation requires hydrolysis of GTP to GDP

Protein Translation 3. Termination
Upon finding a “stop” codon a release factor binds the empty A site The bond between the last amino acid and peptidyl tRNA is hydrolyzed releasing the protein The protein released may not be in its final form Post-translational modification may occur before a protein is fully functional Cleavage Association with other proteins Bonding to carbohydrate or lipid groups

The Polysome (Polyribosome)
Several ribosomes can work together to translate the same mRNA, producing multiple copies of the polypeptide. A strand of mRNA with associated ribosomes is called a POLYSOME (or Polyribosome).

Inhibitors of Protein Synthesis
Two important purposes to biochemists: Inhibitors have helped solve the mechanism of protein synthesis Those that affect PROKARYOTIC but not EUKARYOTIC protein synthesis are effective ANTIBIOTICS Streptomycin: an aminoglycoside antibiotic that induces mRNA misreading; the resulting mutant proteins slow the rate of bacterial growth Puromycin: binds at the A site of both prokaryotic and eukaryotic ribosomes, accepting the peptide chain from the P site, and terminating protein synthesis

Inhibitors of Protein or RNA Synthesis
EFFECT Acting only on Bacteria (Anti-bacterial) Tetracycline blocks binding of aminoacyl-tRNA to A-site of ribosome (30S) Streptomycin prevents the transition from initiation complex to chain-elongating ribosome and also causes miscoding (30S) Chloramphenicol blocks the peptidyl transferase reaction on ribosomes (50S) (Step 2) Erythromycin blocks the translocation reaction on ribosomes (Step 3) Rifamycin blocks initiation of RNA chains by binding to RNA polymerase (prevents RNA synthesis) Acting on Bacteria and Eukaryotes Puromycin causes the premature release of nascent polypeptide chains by its addition to growing chain end Actinomycin D binds to DNA and blocks the movement of RNA polymerase Acting only on Eukaryotes Cycloheximide blocks the translocation reaction on ribosomes (Step 3) Anisomycin blocks the peptidyl transferase reaction on ribosomes (Step 2) α-Amanitin blocks mRNA synthesis by binding preferentially to RNA polymerase II

Puromycin is a Protein Synthesis Inhibitor
Formation of a peptide bond between puromycin at the A site of a ribosome and the nascent peptide bound to the tRNA in the P site. The product of this reaction is bound only weakly in the A site and dissociates from the ribosome, thus terminating protein synthesis and producing an incomplete, inactive peptide.

Chapter 4 DNA, RNA, and the flow of genetic information

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Chapter 4 DNA, RNA, and the flow of genetic information

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