Chapter 11 Part A: Metabolism: The synthesis of nucleic acids and proteins

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1 Chapter 11 Part A: Metabolism: The synthesis of nucleic acids and proteins I. Synthesis of DNA = REPLICATION A. Components of DNA (Fig. 11-1) 1. Composed of 4 different nucleotides that are joined by the phosphate groups to form long polynucleotide chains. A nucleotide is composed of a phosphate group, a deoxyribose sugar, and a nitrogen base. Deoxyribonucleic Acid (DNA) 4 different nucleotides (adenosine, guanosine, thymidine, cytidine) phosphate Deoxyribose sugar Nitrogen bases adenine guanine thymine cytosine = A = G = T = C } Purines } Pyrimidines 2. Chargraff s rules (in each DNA molecule: T+C =A+G and T=A and C=G) B. The structure of DNA (Fig. 11-2) 1. Right-handed double helix 2. Each helix has a series of nucleotides held together with phosphodiester bonds. 3. The helices themselves are held together by hydrogen bond between the nitrogen bases: G pairs with C; A pairs with T (Fig. 11-3) basepairs per turn of the helix. 5. The helices are antiparallel 6. DNA molecule has major and minor grooves 7. DNA molecule usually depicted as an inflexible rod, but it can be bent or curved sometimes. 8. Stabilization forces in DNA are from the hydrogen bonding between bases (GC base pair provides more stability because 3 H bonds vs. 2 H bonds for AT basepair) and the stacking of the bases which excludes water molecules. 1

2 C. Organization of DNA in cells (Fig. 11-4) 1. Prokaryotes a) "nucleoid" b) attached at some point to the membrane c) usually circular DNA d) associated with basic histone-like proteins to become "supercolied" 2. Eukaryotes (Fig. 11-5) a) in nucleus b) linear DNA c) associated with histones to form nucleosomes ("beads on a string") which are further coiled to condense into chromosomes D. DNA synthesis in eubacteria classical semiconservative 1. Semiconservative DNA replication (Fig. 11-7) 2. Direction of synthesis of each new strand is 5 3 (Fig ) 3. Replication is bi-directional (Fig. 11-8) 4. Requirements for DNA replication in eubacteria a) origin of replication (oric) which is a 245 basepair site that contains multiple direct repeats where DNA replication begins b) DnaA (unwinds the DNA strands at oric) c) SSB (single stranded binding protein to keep the DNA strands apart) d) Rep is a helicase that disrupts ( melts or denatures ) the H bonds at the replication fork. e) Primosome (1) Primase (synthesizes RNA primers to start DNA replication) (2) DnaB, DnaT, PriA, PriB, PriC f) DNA polymerase (there are 3 types of DNA polymerase that have been isolated from E. coli DNA poliii is the main one used for replication) g) Deoxyribonucleotides (dntps) to incorporate into the new DNA h) Template DNA i) Ribonucleotides to make primers j) Tus protein helps with termination k) ter = termination sites on the DNA 5. Steps in DNA replication (Fig ) a) Binding of DnaA to oric and initial unwinding of the helix b) RepA helicase melts the DNA at the replication fork c) Priming DNA synthesis Primase synthesizes RNA primers. d) Leading strand DNA synthesis: DNA poliii synthesizes new chains in the 5 to 3 direction. Since the DNA helices are antiparallel, the direction of movement relative to the template DNA strand is 3 to 5. e) Lagging strand is synthesized discontinuously as short fragments (Okasaki fragments). The RNA primers in these fragments are later 2

3 removed by DNA poli, and the fragments are joined together by DNA ligase. f) If leading strand synthesis is going in 1 direction and lagging strand synthesis is going in the other direction, how does 1 DNA poliii dimer synthesize both strands at once????? Looping of the template strand for lagging strand synthesis allows DNA poliii at replication fork to synthesize both the leading and lagging strands simultaneously. g) Rep helicase is continuously melting the DNA at the replication fork. h) Exonuclease editing allows for proofreading of DNA synthesis: (epsilon subunit of DNA poliii and also DNA poli itself) i) DNA gyrase relaxes supercoils that form ahead of the replication fork j) Termination occurs when the pol reaches the termination sites (ter) on the DNA with the aid of the Tus protein E. DNA replication: Rolling circle (Fig. 11-9) 1. In a) some prokaryotes during conjugation b) some phage c) rrna genes in some eukaryotic egg development 2. Mechanism a) circular DNA is nicked b) 3' end of nick is used as primer for DNA synthesis c) 5' end of same strand is displaced during DNA synthesis d) other strand "rolls' and acts as a template e) complementary strand synthesis occurs using displaced strand as template f) end result is concatamer of DNA that can be cleaved if needed F. DNA replication in eukaryotes - Additional considerations 1. Enzymology of DNA replication similar to in eubacteria 2. Frequently, more than one chromosome 3. Complex structure of the chromosomes 4. Much larger amount of DNA 5. Multiple origins (Fig ) 6. 4 DNA polymerases have been identified 3

4 II. Synthesis of RNA = TRANSCRIPTION A. General features of RNA 1. Contains ribose instead of deoxyribose 2. Contains uracil instead of thymine 3. Single stranded instead of double stranded (although there are regions of pairing) 4. Three types a) mrnas carries information for synthesis of a gene product (1) polycistronic (carries information from more than 1 gene) in prokaryotes (Fig ) (2) monocistronic in eukaryotes b) trnas carries specific amino acids during protein synthesis c) rrnas structural components of ribosomes B. Transcription general info 1. Each RNA species is complementary to one strand (template strand) of the DNA double helix. 2. Upstream vs. downstream: RNA strand has a 5 and 3 end. Upstream refers to towards the 5 end and downstream refers to towards the 3 end. 3. The region of DNA that contains sequences that are the signals for transcribing a gene are termed promoters refers to the basepair where transcription starts; -x refers to x basepairs 5 to the start site. C. Factors required for eubacterial transcription 1. RNA polymerase (enzyme that catalyzes the synthesis of RNA from a DNA template). a) Core enzyme = 3 different types of subunits (2α; 1β; 1β ) (1) β - binds incoming nucleotides (2) β binds DNA (3) α - helps with enzyme assembly; interacts with other transcriptional activator proteins; recent work demonstrated that α also interacts with some DNA sequences b) σ factors Initially, people thought that there was only one σ factor that functioned to direct RNAP to the promoters of genes. Later, different classes of σ factors were found. Each σ factor directs RNAP to a different type of promoter (differentiated by a specific DNA sequence in the promoter). c) Holoenzyme = core + σ factor (recognizes the promoter) 2. Accessory transcription activator proteins a) Can bind to specific DNA sequences and help RNA polymerase initiate transcription via protein-protein interactions or by altering the structure of the DNA. b) Transcription of some promoters requires an accessory transcriptional activator; at other promoters, the activators just increase the rate of transcription but are not absolutely required. 4

5 3. Template DNA containing gene or genes to be transcribed 4. Promoter - The regulatory element that determine when a gene turned on (transcribed) or turned off. The promoter DNA is located upstream of the gene and contains a sequence which σ factor of RNAP and other transcription factors bind. Different classes of promoters have different DNA sequences. Deviations from the consensus sequence decrease the level of transcription. Promoter Function -35 sequence spacer -10 sequence Sigma 70-dependent Housekeeping TTGACA TATAAT Sigma 32-dependent Heat shock stress response TCTCNCCCTTGAA CCCCATNTA Sigma 28-dependent Flagella synthesis CTAAA CCGATAT Sigma S-dependent Stationary phase survival?? Sigma 54-dependent Nitrogen utilization; pilin CTGGNA (-24) TTGCA (-12) 5. Weak promoters (ones that have poor sigma recognition sequences) have additional sequences to which transcriptional activators can bind. 6. NTPs, Mg Transcriptional terminator (Fig ) D. Factors required for eukaryotic transcription 1. RNA polymerases Much more complex that prokaryotic RNAP (numerous additional factors required, multiple polymerases) a) RNAP I synthesizes rrna b) RNAP II synthesizes mrna c) RNAP III synthesizes trna and 1 type of rrna 2. Eukaryotic RNAPs have subunits that are homologous to α, β, and β of prokaryotic RNAP; however, eukaryotic RNAP also contain many additional subunits 3. Eukaryotic promoters contain some combination of the following a) contain a TATA rich region located 25 to -30 from the start of transcription b) Upstream from the TATA region is a variably located sequence containing the sequence CCAAT (frequently at 75) c) GC box (frequently at 90) d) Some promoters have other sequences located either upstream or downstream that maximize the level of transcription called enhancers E. Eubacterial transcription (see attached) 1. Initiation a) RNAP scans the DNA looking for promoters b) σ factor of RNAP binds the corresponding σ factor recognition sequence in the promoter. c) Recent evidence suggests that at some promoters, the α subunit may bind to AT rich regions upstream of the sigma binding sites. 5

6 d) RNAP is bound covering approx. 60 basepairs. The DNA is still is a double helix (closed complex). e) RNAP unwinds the DNA resulting in open complex formation f) First nucleotides are added to start RNA chain. Transcriptional initiation has occurred! g) Accessory transcription factors may aid in all of the above listed steps. 2. Elongation a) Elongation is 5 3 b) σ factor is ejected from RNAP after first 2-10 nucleotides are added. c) Much less is known about this step for transcription than initiation. It was once believed that elongation occurred at a constant rate; however, recent work suggests that RNAP may pause during elongation. In fact, pausing is important in termination (see below). 3. Termination (2 types) a) Rho independent: A specific sequence at the end of the gene signals termination. The sequence is transcribed into RNA and it is the RNA sequence that is important. This sequence contains numerous Gs and Cs, which forms a hairpin structure, followed by a string of Us. The hairpin destabilizes the DNA:RNA hybrid leading to dissociation of the RNA from the DNA. b) Rho dependent: Rho protein binds to a sequence in the RNA (rut site not well characterized). Rho moves along the RNA in the 3 direction until in eventually unwinds the DNA:RNA hybrid in the active site, thereby pulling the RNA away from the DNA and RNAP. Rut sites are located 5 to sites in the DNA that cause RNAP to pause. It is thought that this allows Rho to catch up to RNAP and the RNA-DNA hybrid. F. Eukaryotic transcription (Fig ) Initiation and elongation are similar to in prokaryotes; however, there are several important differences. 1. In prokaryotes, mrna is translated into protein as transcription is occurring, while in eukaryotes the mrna is transported to the cytoplasm before translation occurs capping in eukaryotes a) Guanosyltransferase adds 5 methyguanosine (Cap) to 5 end of mrna early in transcription (Fig ) b) The Cap is important for translation initiation and for export from the nucleus poly(a) tail in eukaryotes a) AAUAAA sequence in the RNA signals a cleavage event in the RNA by endonuclease. b) Poly(A) polymerase then adds A residues to the 3 end of the mrna. c) The poly(a) tail increases the stability of the mrna in eukaryotes. 6

7 d) As a side note, recent evidence has demonstrated that there are poly(a) polymerases in prokaryotes and that some mrnas have poly(a) tails. Interestingly though, the polya tail destabilizes the mrna in prokaryotes. 4. Splicing of primary transcript In most prokaryotes, genes are contiguous segments of DNA; while in eukaryotes genes are often interrupted by noncoding DNA (introns). a) The primary transcripts often contain intervening sequences (introns) that are removed from the RNA prior to translation by a cleavage reaction catalyzed by snrnps (small nuclear ribonuclear proteins which contain RNA and protein). b) Frequently, the splicing site in the intron has a GU at the 5 end and an AG at the 3 end. The snrnp aligns these ends in a lariat formation to allow precise splicing. c) Complexes containing the snrnp, mrna, and associated proteins are called spliceosomes. d) Splicing is important (1) splicing allows variations of a gene and therefore gene product to be made (2) it has been suggested that exons correspond to functional motifs in proteins and thus the presence of genes that require slicing allows for evolutionary tinkering (3) many viruses have spliced mrnas and so understanding the process may lead to new therapeutic approaches. 7

DNA REPLICATION. Third Stage. Lec. 12 DNA Replication. Lecture No.: 12. A. Watson & Crick (1952) C. Cairns (1963) autoradiographic experiment

DNA REPLICATION. Third Stage. Lec. 12 DNA Replication. Lecture No.: 12. A. Watson & Crick (1952) C. Cairns (1963) autoradiographic experiment Lec. 12 DNA Replication A. Watson & Crick (1952) Proposed a model where hydrogen bonds break, the two strands separate, and DNA synthesis occurs semi-conservatively in the same net direction. While a straightforward