Delve AP Biology Lecture 7: 10/30/11 Melissa Ko and Anne Huang

Transcription

1 Today s Agenda: I. DNA Structure II. DNA Replication III. DNA Proofreading and Repair IV. The Central Dogma V. Transcription VI. Post-transcriptional Modifications Delve AP Biology Lecture 7: 10/30/11 Melissa Ko and Anne Huang I. DNA Structure In eukaryotes, the genome is usually much larger [for example, prokaryotes only have one circular chromosome, while eukaryotes can have many linear chromosomes]. Since the eukaryotic genome is larger, it needs to be highly organized and condensed [otherwise it wouldn t be able to fit in the nucleus. If you stretch out the entire human genome, it would be around 3m or 9ft long!] DNA is wrapped around histone proteins. This DNA-histone complex is called chromatin. Chromosomes are highly condensed chromatin. Chromosomes are formed only during cell division, when the cell needs to divide its genome evenly between the two daughter cells. You can see chromosomes, but you can t see chromatin. When people were first observing cells under microscopes, they saw these little X s in the nucleus and they didn t know what they were, so they called them chromosomes [Greek for colored bodies ]. Not all of the DNA is at the same level of packing at the same time. Heterochromatin is more tightly packed chromatin, while euchromatin is less compact. Different levels of packing can influence gene expression. Genes in less compact chromatin are more likely to be expressed because 4 levels of structure [simplified version of structure found in archea] 1. Nucleosomes [10-nm fiber], looks like beads on a string In chromatin, the mass of histones is equal to the mass of DNA Histones are positively-charged, while DNA is negatively-charged from the phosphate groups in the phosphate-sugar backbone Each nucleosome is made of 8 histones, the amino ends stick out to form tails The DNA between each nucleosome is called linker DNA, people currently believe that linker DNA does not code for any protein 2. Nucleosomes pack together [30-nm fiber] 30-nm refers to the thickness of the fiber 3. Looped domains [300-nm fiber] The 30-nm fibers form loops on a protein scaffold [not histones] 4. Chromosome [ONLY DURING CELL DIVISION] 1

2 Looped domains coil and fold to form chromosomes II. DNA Replication After Watson and Crick determined the structure of DNA, they realized that DNA replication probably had something to do with complementary base-pairing. If there is one template strand, a complementary strand will assemble on its own due to the A-T, G- C base pairing. Modes of Replication Conservative: The parent DNA dissociates, and each strand acts as template to form two new DNA strands. The parent DNA strands then reassociate and new DNA strands associate with each other. Semi-conservative: The parent DNA dissociates, and each strand acts as template to form two new DNA strands. The resulting daughter DNA consists of one strand of parent DNA and one newly synthesized strand. Dispersive: Each strand of new DNA contains a mixture of old and new DNA Meselson-Stahl experiment [late 1950s] The Meselson-Stahl experiment proved that DNA replicates semi-conservatively. 1. Grow E. coli on a medium with radioactively labeled nucleotides ( 15 N, a heavy isotope of nitrogen) 2. Transfer E. coli to a medium with nucleotides labeled with a lighter isotope of N ( 14 N) 3. After one round of cell replication, they extracted the DNA and centrifuged it. They saw that the new DNA formed a single band was in between 15 N and 14 N, which disproved the conservative mode of replication. 4. Then, they allowed cells to undergo two rounds of replication before extracting and centrifuging the DNA. They saw two bands: one was in between 15 N and 14 N, and the other was 14 N, disproving the dispersive mode of replication DNA replication DNA replication begins at the origin of replication. There is only one origin in prokaryotes, while there are many in eukaryotes. 1. Helicase unwinds double helix. Unwinding causes tighter twisting, so topoisomerase breaks and rejoins DNA to relieve the extra strain. Single-strand binding proteins bind to the single-stranded DNA and stabilize it 2. Initiation of elongation: First, you need primase to add an RNA primer [an short nucleotide chain]. We need a primer before elongation can occur because DNA polymerase can only add nucleotides to the 3 end of an existing strand. It cannot begin a new DNA strand on its own. 3. Elongation: DNA polymerase III catalyzes the addition of a complementary nucleotide. It takes a nucleoside triphosphate and hydrolyzes it to release energy. [A nucleoside triphosphate is a nucleotide with 3 phosphate groups instead of just 1. It looks like ATP except that it has a deoxyribose instead of a ribose sugar]. The nucleoside triphosphate loses 2 phosphate groups, and a phosphodiester bond is formed from the 3 OH and the 5 phosphate. DNA POL ALWAYS SYNTHESIZES 5 TO 3. 2

3 4. Leading and lagging strands. The lagging strand has several RNA primers. DNA polymerase III elongates until it meets a primer. These DNA fragments are called Okazaki fragments. 5. We can t leave RNA primers in newly synthesized DNA, so DNA polymerase I replaces RNA primer with DNA nucleotides. 6. Finally, DNA ligase catalyzes the formation of phosphodiester bonds to connect the Okazaki fragments. Trombone model: Campbell refers to this as a stationary complex, a DNA replication machine III. DNA Proofreading and Repairing It is important that DNA replication is highly accurate, because we don t want mutations to accumulate. The error rate is estimated to be 1 in 10 billion nucleotides. Proofreading: As soon as DNA polymerase adds a new nucleotide, it checks it with the template. If the newly added nucleotide is incorrect, DNA polymerase removes the nucleotide and resumes elongation. Repair: If DNA polymerase missed an incorrect nucleotide while proofreading, it is not too late to correct the mismatch! The cell is continuously monitoring and repairing its DNA to minimize mutations. Mismatch repair: A specific kind of repair is called nucleotide excision repair, where nuclease cuts out [excises] incorrect DNA. DNA polymerase I fills in the missing nucleotides, and DNA ligase reconnects the new DNA with the existing strand. IV. The Central Dogma Beadle and Tatum: one gene-one polypeptide hypothesis DNA is transcribed to mrna, which is translated to protein This hypothesis is a bit simplified. While it is usually true, some genes encode for RNA molecules that have important functions and are not translated into protein. Also, sometimes RNA can be reverse transcribed into DNA [ex: viruses like HIV] Why have an mrna intermediate? 1. Protects DNA: If something happened to the DNA while making it into protein, it would be very bad for the cell 2. Making multiple RNA copies means that the cell can translate many copies into protein simultaneously Transcription and translation in prokaryotes is slightly different from transcription and translation in eukaryotes. For instance, transcription and translation can be continuous in prokaryotes because there is no nucleus separating the DNA from the ribosomes. 3

4 V. Transcription Divided into initiation, elongation, and termination Initiation Promoter: region of DNA where transcription begins. The promoter also indicates which strand is the template strand. In eukaryotes, there is a part of the promoter that contains a lot of Ts and As. This is called a TATA box RNA polymerase binds to the promoter. In prokaryotes, RNA polymerase itself recognizes and binds to the promoter. In eukaryotes, transcription factors help RNA polymerase and the promoter bind together, forming a transcription initiation complex. The TATA box is very important in forming the transcription initiation complex. Once RNA polymerase binds, the DNA strands unwind. Elongation As RNA polymerase travels down the DNA, it unwinds the DNA by itself [it doesn t need another enzyme like helicase, like in DNA replication]. RNA polymerase reads off the template strand and adds complementary RNA nucleotides **RNA POL ALWAYS SYNTHESIZES 5 TO 3 Termination In prokaryotes, there is a terminator, which is a DNA sequence that signals the end of transcription. The terminator causes RNA polymerase to fall off the DNA and release the mrna transcript. This newly transcribed mrna can be immediately translated into protein. In eukaryotes, it is more complicated. There is a polyadenylation signal sequence, or a region of DNA [TTATT]. RNA polymerase transcribes it and there is a repeating sequence of AAUAA. Proteins recognize this polyadenylation signal and cut the growing pre-mrna strand. The pre-mrna is floating free and RNA polymerase continues transcribing the DNA. RNA polymerase eventually falls off DNA [but the mechanism is still unknown]. VI. Post-transcriptional Modifications [EUKARYOTES ONLY] In eukaryotes, the newly synthesized mrna still needs to be modified before it can be transported out of the nucleus and translated. 5 cap: Add a modified form of guanine nucleotide to 5 end 3 poly A tail: Add lots of adenine nucleotides to 3 end [50-250] Neither the 5 G cap nor the 3 poly A tail are translated in protein Why modify the ends of mrna? May promote export of mrna from nucleus to cytoplasm May stabilize mrna and prevent it from degrading Helps ribosome attach at the right end [5 ] to begin translation 4

5 RNA splicing In a eukaryotic gene, there are noncoding regions of DNA called introns that are sandwiched between coding regions called exons [introns do not exist in prokaryotes!]. Both introns and exons are transcribed by RNA polymerase, but the introns need to be spliced out and the exons need to be joined together before mrna can be translated. At the end of each intron, there is a short DNA sequence which signals where splicing should occur. snrnps [small nuclear ribonucleoproteins] recognize these sequences. As the name implies, snrnps are small molecules made of RNA and protein, located in the nucleus. Several different snrnps join together to form a larger assembly called a spliceosome, which cuts out the introns [example of ribozyme]. Why do eukaryotes have introns? People think that introns may be involved in gene regulation Alternative RNA splicing: can get several different polypeptides from the same gene 5