Cell Biology, Final Exam - Ultrastructure of Replication and Transcription

List the role of the three eukaryotic RNA polymerases. - Basic Info - How do they work? - Prokaryote vs. Eukaryote - Give 3 Main (with function)

RNA Polymerase - General: a. Any group of enzymes that catalyze the synthesis of RNA using DNA as a template. b. They function by adding successive nucleotides to the 3' end of the growing RNA strand. c. Bacteria have one primary RNA polymerase but in eukaryotes the labor is subdivided. RNA Polymerase - 3 Main: 1. RNA POLYMERASE I (Pol I) - A type of eukaryotic RNA polymerase present in the nucleolus that synthesizes an RNA precursor for three of the four types of rRNA. a. 5.8S rRNA b. 18S rRNA c. 28S rRNA 2. RNA POLYMERASE II (Pol II) - A type of eukaryotic RNA polymerase present in the nucleoplasm that synthesizes pre-mRNA and most of the snRNAs. - snRNAs are small nuclear RNAs involved in posttranscriptional RNA processing. This RNA polymerase is responsible for the greatest variety of RNA molecules. 3. RNA POLYMERASE III (Pol III) - A type of eukaryotic RNA polymerase present in the nucleoplasm that synthesizes a variety of small RNAs, including pre-tRNAs and 5S rRNA.

Describe the replication of bacterial and T7 viral DNA as determined by autoradiography and electron microscopy. - What does this visualization show?

T7 Viral DNA Replication: a. Begins at an origin and proceeds in a bidirectional fashion. b. Microscopy: - Seen in an EM of a chromosome that is labeled with a low level of irradiated thymine. - Then, when replication begins, they are exposed to a higher level of tritium. - The higher level of tridiated thymine is visible in both forks. - This indicates that BOTH forks are being replicated at the same time.

Briefly discuss the splicing of mRNA.

a. This mechanism is so highly conserved that it is universal. b. Mammalian mRNA can be spliced in yeast. c. The spliceosome recognizes sequences on the messenger at the intron junctions, cuts the introns out and ligates the exons together. d. This is often done because you can use one gene to make many proteins using different splicing patterns. Ex: a. The initial transcript of a gene can be 7,700 bp long. b. Has its 5' and 3' ends modified via capping and polyadenylation. c. The transcription is then spliced, leaving a mRNA of 1,872 base pairs. - About ¾ of the transcript of this gene is removed.

Compare hnRNA to mature cytoplasmic mRNA.

a. mRNA that is originally transcribed in the nucleus has been referred to as heterogeneous RNA or hnRNA particles. b. These are the initial transcripts. c. They have not been spliced (if splicing is necessary for a given mRNA). d. Usually quite large compared to the mRNA that is finally derived from it. - The large hnRNAs (the pre-mRNA) are MUCH larger than the nature mRNAs. e. 90% of the pre-mRNA transcripts never makes it out of the nucleus. - That 90% is introns - it is all spliced out.

Describe the transcription of nucleolar DNA (rRNA transcription). 1. rRNA Basics 2. Nucleolar DNA (rRNA) Transcription

rRNA Basics: a. rRNA is produced in the nucleolus. b. As it is transcribed, it is immediately folded up and packaged with proteins. c. rRNA represents about 70-80% of the total cellular RNA. Nucleolar DNA (rRNA) Transcription: a. RNA Polymerase I transcribes the pre-rRNA transcription unit (a sequence of DNA that codes for the three (28S, 18S and 5.8S)) into pre-rRNA. - NOTE: This is a single sequence that codes for ALL three together. - This is because of the large quantities of these rRNAs used in the cell. - This also ensures that they are produced in equal quantities. b. Pre-rRNA is processed by a series of cleavage reactions that remove the transcribed spacers and release the mature rRNAs. c. Otherwise the procedure follows the standard transcription overview.

Describe the Watson Crick model of DNA and its mode of replication. - Give general DNA and replication information. - Give 6 main steps of replication.

DNA: a. The macromolecule that serves as the repository of genetic information in all cells. b. Constructed from nucleotides consisting of deoxyribose phosphate linked to base. c. It forms a double helix held together by complementary base pairing between adenine and thymine (2 bonds), and between cytosine and guanine (three bonds). d. They found b DNA; the main form of DNA in cells. e. There are two other types: A DNA and Z DNA. - A DNA is also a right handed double helix but it is shorter and thicker than B DNA. - Z DNA is a left handed double helix that derives its name from the zigzag pattern of its longer, thinner sugar-phosphate backbone. Replication - General: a. DNA must have a primer to begin. b. There has to be a double stranded region with a 3' hydroxyl sticking out. c. The polymerase must have a primer to attach to. - This primer is an RNA that is laid down by a unique RNA polymerase (primase) and then the DNA polymerase then elongates that to begin replication. d. There are also helixases that unwind the DNA. e. Topoisomerases that control the supercoiling. f. The DNA that runs 5' to 3' is replicated in a non stop fashion because that is the only direction that the polymerase can travel. - The 3' to 5' strand (the lagging strand) is being copied in the 5' to 3' directions in short fragments (Okazaki fragments) that are then pieced together. - Thus the lagging strand requires a series of RNA primers. Replication - 6 Main Steps: 1. The initiator protein binds to the double-stranded DNA at the origin of replication and, using ATP energy, slightly unwinds the DNA. 2. Helicase unwinds the helixes while topoisomerase unwinds the supercoils of the DNA. A replication fork is now in evidence. 3. Primase binds to the first priming sequence on the leading-strand template and synthesizes a short RNA primer that is complementary to the DNA template. 4. DNA polymerase uses the primer to initiate DNA synthesis by adding deoxyribonucleotides to its 3' end. - The new strand that runs in the 5' to 3' end is called the leading strand and it only requires one priming event. 5. An RNA primer is now made for the lagging strand and DNA polymerase will extend the strand with deoxyribonucleotides. 6. For the lagging strand, a series of priming events is required, each generating an Okazaki fragment. - These Okazaki fragments are then linked together by DNA ligase creating a complete strand of DNA.

Discuss the role and mechanism of chromatin remodeling in gene expression.

Histones modification can vary throughout the cell cycle. In terms of regulating genes and transcription, we now know that transcriptional regulation is more than just repressors or activators binding up promoter sites and turning a gene on/off. In eukaryotes, the accessibility of a promoter to the regulator proteins is a consideration. Normally your genes are wrapped around nucleosomes that are then packed together to form an interphase 300A fiber. This keeps the activators and repressors from having access to the DNA. By modifying the histone tails by acetylation, phosphorylation or methylation, we change the ionic characteristics of those tails and therefore change the way they interact with the DNA; the interactions are disrupted and things are loosened up. If a region is going to be transcribed, the chromatin is remodeled into this looser structure. Now the RNA polymerase is able to access the promoter region. Once the DNA is transcribed, the histones have their tails removed and everything gets folded back up.

Discuss the transcription of Balbiani Ring 2 in Chironomus.

Transcription of Balbiani Ring 2 in Chironomus: a. These puffs occur in insect cells with polytene chromosomes. b. The chromosomes continually replicate even though the cells aren't dividing. c. These chromatids line up to form the polytene chromosomes (have characteristic dark pattern bands) d. Activation of the genes of a given band cause the band to uncoil and expand outward resulting in a chromosome "puff" (aka Balbiani rings named after the Italian microscopist that first observed these puffs). e. Using tritiated uracil being incorporated into RNA, we can see the bands on chromosome 4 in Chironomus (a fly related to the fruit fly) by visualizing the silver grains. f. In other words the characteristic puffing patterns seen are direct visual manifestations of the selective decondensation and transcription of specific DNA segments.

Describe the replication of DNA in eucaryotes and explain the role of replicons.

a. Eukaryotic chromosomes are so large that in order to replicate them in a reasonable length of time, they must have multiple origins. b. The replication then proceeds in a bidirectional fashion just as in the prokaryote version. c. Replication seems to be tied to the nuclear skeleton. - The replicative regions are associated with the nuclear skeleton so the nuclear apparatus stays put and the DNA is fed through it and replicated as it goes. d. Replications rates can depend on the age of the organism. - For example: in fruit fly embryos, replication is completed in about 5 minutes, (a bunch of origins being used). In adults, the euchromatin takes about 1 hour (fewer origins being utilized). - How this control is applied is currently unknown. e. Replicons: - The total length of DNA replicated from a single origin of replication. - Multiple per chromosome in eukaryote. - Replicon of prokaryotes is usually in entire chromosome.

Explain the segregation of parental histones (nucleosomes) during replication.

a. In eukaryotes, the DNA must be unwound from the nucleosomes during replication. b. When the DNA is unwound, the histone core doesn't dissociate completely, part of it stays attached to DNA. c. When the DNA is copied and rewound the old and new histones get mixed and matched.