Unit 3 vocab

is a 5' to 3' polymerase

-DNA polymerase I and III and primase

What evidence do we have that, in the helical form of the DNA molecule, the base pairs are composed of one purine and one pyrimidine?

1. Chargaff's rule: A=T and G=C via quantitative analysis 2. X-ray diffraction studies compared the known sizes of the base with the observed dimension s of the DNA double helix.

What exactly is a deoxyribonucleotide made up of, and how many different deoxyribonucleotides are there in DNA? Describe the structure of DNA, and describe the bonding mechanism of the molecule (i.e., the kind of bonds on the sides of the "ladder" and the kind of bonds holding the two complementary strands together). Base pairing in DNA consists of purine-pyrimidine pairs, so why is it not possible for A-C and G-T pairs to form?

2' deoxyribose plus a phosphate group attached to its 5-carbon plus a nitrogenous base attached to its 1' carbon. dAMP, TMP, dGMP, and dCMP. Weaker hydorgen bonds between A-T and G-C. (A+T) cannot equal (G+C)

DNA ligase

A linking enzyme essential for DNA replication; catalyzes the covalent bonding of the 3' end of a new DNA fragment to the 5' end of a growing chain.

Describe the bonding properties of G-C and T-A. Which base pair would be harder to break apart? Why?

A-T--> two hydrogen bonds while G-C has three hydrogen bonds which will be harder to break.

Unwinds double helix

DNA helicase

has 5' to 3' exonuclease activity

DNA polymerase I

Has a 3' to 5' exonuclease activity

DNA polymerase I, II, and III

How is the mechanism of group I intron removal different from the mechanism used to remove the introns in most eukaryotic mRNAs? Speculate as to why these different mechanisms for intron removal might have evolved and how each might be advantageous to a eukaryotic cell.

Group I introns are self-splicing. Unlike removal of introns from most eukaryotic mRNAs, removal of group I introns don't need a spliceosome containing snRNAs and protein splicing factors; group I introns fold in protein-independent manner to catalyze the splicing reaction. Spliceosome processing of introns may also allow for greater diversity in intron size. They can also be regulated using protein factors. Allows for additional level of control over gene expression.

Separate daughter molecules and causes supercoiling

Gyrase

8 Kornberg isolated DNA polymerase I from E. coli. What is the function of the enzyme in DNA replication?

Has the 5' to 3' exonuclease activity of DNA polymerase I; removes RNA primer synthesized during initiation of DNA replication and replace this RNA with DNA. When DNA polymerase III, main synthetic enzyme for DNA polymerization, reaches a RNA primer, it dissociates from DNA. DNA polymerase I functions to continue synthesis of DNA in a 5' to 3' direction. Simultaneously removes the RNA primer using the 5' to 3' exonuclease activity and replaces RNA with DNA nucleotides.

Distinguish between the actions of helicase and topoisomerase on double-stranded DNA and their roles during DNA replication.

Helicase untwists two strands to separate them while topoisomerase alters the tension in a dsDNA molecules introduced by supercoiling. When helicase untwists the two strands in dsDNA during DNA replication, tension is produced ahead of the replication fork due to supercoiling in that region. Topoisomerase add or remove negative supercoils from cellular DNA. During DNA replication, the topoisomerase DNA gyrase relaxes the tension ahead of the replication fork.

Histone proteins from many different eukaryotes are highly similar in their amino acid sequence, making them among the most highly conserved eukaryotic proteins. What functional properties of histone proteins might limit their diversity?

Histones perform the same basic role in organizing and packaging DNA in chromosomes of all eukaryotes. When five histones form nucleosome complexes on DNA, the histones interact with all types of DNA sequences- they don't physically interact with DNA in a sequence specific manner. Means that histones must be able to interact with each other and form a complex that recognizes DNA features common to all dsDNA within the DNA double helix such as spacing of the nucleotides and location and charge distributions on sugar and phosphate residues. They also must be able to complex DNA in a manner that allows it to form orderly structures that compact DNA and enables themselves to carry out further packaging through interactions with nonhistone scaffold.

How could you use radioactively labeled molecules to determine if the genome of a newly identified bacteriophage that infects E. coli is RNA or DNA? How might you determine if it is composed of single-stranded or doublestranded nucleic acid?

If its DNA, infect and label with dTTP and collect progeny phage. If its RNA, infect with UTP. To see if its double strand or single strand, determine the composition and evaluate whether the molar ratio of A and T(U) match and G and C match- indicates double strand.

Primary Effects of migration

Increase genetic variation, and has the potential to disrupt a Hardy-Weinberg equilibrium. It can increase genetic variation and may influence the evolution of allele frequencies within populations. Over many generations, migration will reduce divergence among populations and equalize allele frequencies among populations.

List the components necessary to make DNA in vitro, using the enzyme system isolated by Kornberg.

Kornberg enzymes (DNA polymerase I): all four dNTPs (dATP, dGTP, dCTP, dTTP), magnesium ions and a fragment of double stranded DNA that will serve as a template.

Bonds the free 3' -OH end of a polynucleotide to a free 5' monophosphate end of a polynucleotide

Ligase

Primary effects of mutation

Mutation will lead to change in allele frequencies within a population if no other forces are acting. Introduces genetic variation. If effective population size is small, mutation may lead to genetic differentiation among populations

What is the relationship between cellular DNA content and the structural or organizational complexity of the organism?

No simple relationship between haploid DNA content of a cell and its structural or organization complexity. Called the C-value paradox; organisms in some taxa show little variation, organisms in other taxa show as much as tenfold in variation in their C-values. Considerable variation in amount of repetitive-sequence DNA in the genome.

Discuss the components and structure of a nucleosome and the composition of a nucleosome core particle. Explain how nucleosomes are used to package DNA hierarchically

Nucleosomes are fundamental unit of DNA packaging in eukaryotic chromosomes. In a nucleosome core particle, short segment of DNA is wrapped about 1 1/4 times around a protein core. Protein core is an octamer consisting of two copies of each of the four histones: H2A, H2B, H3, H4. Nucleosome core particle packs the DNA into a flatten disk about 5.7 x 11 nm. Packaging into nucleosomes effectively condense DNA by a factor of 7. Interactions between the histones and DNA are not sequence specific, but rather are based on the basic, positively charged histone proteins interacting with the acidic, negatively charged sugar-phosphate backbone of the DNA. Nucleosome core particles serve to package double stranded DNA at regular intervals with short stretches of linker DNA between particles. At this level of packaging, DNA is packed like beads on a string form of chromatin known as 10 nm nucleofilament. Condensation of DNA also forms the basis for subsequent packaging, first through associations betwene nucleosomes and then by associations between nonhistone chromosomal proteins. Associations between nucleosomes result in further compaction of the 10 nm filament, producing a 30 nm chromatin fiber that condenses DNA another sixfold. Histone H1 plays an important role in this coiling. The 30 nm chromatin fiber is further condensed into looped domains, with each loop containing 10 to 100 kilobases of DNA. The looped domains are anchored inside the nuclear envelope to a filamentous structural framework of protein, the nuclear matrix.

How overdominance leads to increase in sickle cell anemia

Overdominance results when a heterozygote genotype has higher fitness than do either of the homozygotes. The two alleles of the heterozygotes are maintained in a population because both are favored in heterozygote genotype.

Is a DNA polymerase

Polymerase I and III

Bonds the 3'-OH end of a polynucleotide to a free 5' nucleotide triphosphate

Polymerase I, III, and primase after the first two bases of RNA primer are positioned

is the major elongation enzyme

Polymerase III

Which of the mutations that follow are likely to be recessive lethal mutations (i.e., mutations causing lethality when they are the only alleles present in a homozygous individual) in humans? Explain your reasoning. a. deletion of the U1 genes b. a single base-substitution mutation in the U1 gene that prevented U1 snRNP from binding to the 5'-GU-3' sequence found at the 5' splice junctions of introns c. deletion within intron 2 of beta-globin d. deletion of four bases at the end of intron 2 and three bases at the beginning of exon 3 in beta-globin

Recessive lethal mutations result in death because some essential function is lacking. Neither copy of gene functions. a. U1 deleted will be recessive lethal, as U1 snRNA is needed for identifying the 5' splice site in RNA splicing. Incorrect splicing would lead to nonfunctional gene products for many genes, a nonviable situation. b. recessive lethal because prevents base pairing with 5' splice sites c. If the deletion doesn't affect the branchpoint, then its fine. If it does, could result in production of nonfunctional hemoglobin protein if homozygous. d. Deletion would at best make an aberrant splicing of that intron. If homozygous, only nonfunctional protein is produced.

Prevents reassociation of complementary bases

SSB protein

What are the most significant differences between the organization and expression of bacterial genes and eukaryotic genes?

Structure: Prokaryotic genes have an upstream promotor, an RNA-coding sequence and a downstream terminator. While these three features also exist in eukaryotic genes, eukaryotic promoters are more complex, and nearby or distant enhancer and silencer elements can strongly affect the level of transcription of eukaryotic genes. The RNA-coding sequences of eukaryotes can be interrupted with introns. Finally, prokaryotic mRNAs are often polycistronic, containing the amino acid coding information for more than one gene. In contrast, eukaryotic mRNA are generally monocistronic, containing the amino acid coding information from just one gene. A notable exception is C.elegans where polycistronic mRNAs are found at central loci. Processing: Prokaryotic genes lack introns, while eukaryotic genes have one or more introns; therefore a transcribed region can be larger than the size of a mature mRNA. Excision of introns from primary mRNAs is only one aspect of processing for eukaryotic RNAs; they also modified at both their 5' to 3' ends where they are capped an polyadenylated. Coupling of Transcription and translation: Since prokaryotes lack a nucleus, transcription is directly coupled to translation. IN eukaryotes, mRNAs must be processed then transported out of the nucleus before translation in the cytoplasm. Give three means to regulate eukaryotes.

Telomeres are unique repeated sequences. Where on the DNA strand are they found? Do they serve a function?

Telomeres are characteristically heterochromatic sequences found at the ends of a linear eukaryotic chromosome. For most organisms, the telomeric sequences at the extreme end of the chromosome are simple, highly repeated, and species specific. Nearby, not at the very end of the chromosome, are telomere associated sequences which are repeated complex sequences and extend for thousands of bases form the simple telomeric sequences. These sequences are replicated, along with other chromosomal DNA sequences by a set of enzymes that include DNA polymerases. Telomere organization is quite different from others. Telomeres function in DNA replication and provide chromosome stability.

Define topoisomerases, and list the functions of these enzymes.

Topoisomerase is a class of enzymes that convert one topological form of DNA to another. They untwist relaxed DNA to produce negative supercoils or twist negatively supercoiled DNA to convert it to a more relaxed state. These enzymes allow DNA to be converted between negatively supercoiled, compact DNA and a more relaxed, less compact state.

Suppose you identify a previously unknown multicellular organism. a. What composition do you expect its genome to have? b. How would your answer change if it were a unicellular organism? c. How would your answer change if it were a bacteriophage or virus? d. Do your answers offer any insights into the origins of cellular organisms?

a,b,c. All known cellular organisms use dsDNA so newly discovered multi-cellular or unicellular organisms are expected to have dsDNA genomes. In contrast, variety of bacteriophage and viral genomes are known. They can be single or double stranded DNA or RNA d. No.

Hershey and Chase showed that when phages were labeled with 35S 35S, 32P and the remained outside the cell and could be removed without affecting the course of infection, whereas the 32P entered the cell and could be recovered in progeny phages. a. What distribution of isotopes would you expect to see if parental phages were labeled with isotopes of i. C? ii. N? iii. H? b. Based on your answer, explain why Hershey and Chase used isotopes of phosphorus and sulfur in their experiments.

a. In each case, page ghosts and progeny would be labeled with isotope. Both amino acids and nucleic acids have C, N, and H, so parental phage labeled with isotopes of C, N, and H will have labeled protein coats and DNA. Isotope would be recovered in the DNA of the progeny Phages as well as the phage ghosts left behind in the supernatant after phage infection. b. Isotypes of phosphorous selectively label DNA while isotypes of sulfur selectively label protein. The selective labels allow hershey and chase to track the protein and nucleic acid components of the phage as they reproduced. By selectively labeling DNA and protein, Hershey and Chase could distinguish between DNA and protein as genetic material.

4 The double-helix model of DNA, as suggested by Watson and Crick, was based on DNA data gathered by other researchers. The facts fell into the following two general categories: a. chemical composition b. physical structure

a. Made up of polynucleotides A,T,G,C and follow Chargaff's Rules where A=T and G=C. % GC varies but ratios of A/T and G/C don't change. b. Structure composed of a sugar, base and phosphate. Highly ordered and helical. There are two distinctive regularities at 0.34 and 3.4nm along the molecules axis.

When the eukaryotic chromosome duplicates, the nucleosome structures must duplicate. a. How is the synthesis of histones related to the cell cycle? b. One possibility for the assembly of new nucleosomes on replicated DNA is that it is semiconservative. That is, parental nucleosomes are assembled on one daughter double helix and newly synthesized nucleosomes are synthesized on the other daughter double helix. Is this what happens? If not, what does occur?

a. Most new histone synthesis occurs during the S stage of the cell cycle and is coordinated with DNA replication. b. New nucleosomes are not replicated semiconservatively. Nucleosomes disassemble from DNA as the replication fork passes a replicating DNA region and then are reassembled on the two DNA double helices past the replication fork using components of both old and new histones. Histone chaperone proteins direct the process of nucleosome assembly.

Primary effect of genetic drift

changes in allele frequencies within a population. Can reduce genetic variation and increase homozygosity within a population. Over time, it leads to genetic change. When several populations are compared, genetic drift can lead to increased genetic differences among populations

RNA world hypothesis

hypothesis that RNA served as the genetic information of early life

Primary effect of inbreeding

increases the homozygosity within a population and decreases genetic variation.

Is the "repair" Enzyme

polymerase 1

Is an RNA polymerase

primase

What causes genetic drift?

random changes in allele frequency due to chance. Random factors producing mortality in natural populations and sampling error can lead to genetic drift. Causes a small effective population size over many generations, small number of founders (founders effect), and a reduction in population size (bottleneck effect)

gRNAs

short RNAs partially complementary to regions of the pre-edited mRNA. Found in nucleus and mitochondria. Involved in RNA editing, posttranscriptional insertion or deletion of nucleotides or the modification of one base to another. In trypanosomes where RNA editing results in the removal and insertion of U nucleotides, the gRNA pairs with mRNA transcript and is thought to be responsible for cleaving the transcipt, templating the missing U nucleotides, and ligating the transcript back together again.

snRNAs

small nuclear RNAs, found in the nucleus and used in RNA splicing. They form snRNPs by complexing with proteins. snRNPs assemble around intronic sequences to form spliceosomes to remove the intronic sequences and splice together exonic sequences. Highly abundant with at least 10^5 copies per cell. Their abundance reflects the large number of transcripts with introns that must be processed before the transcripts are exported from nucleus to cytoplasm for translation