Cell and Molec. Biology Exam 2

Describe the structure of a DNA nucleotide?

• Pentose, deoxyribose sugar

• Phosphate group attached to the 5’ carbon on sugar

• Nitrogenous base attached to the 1’ carbon on sugar

• The pentose and phosphate group together compose the sugar-phosphate backbone of the DNA helix

How are nucleotide subunits joined together?

Via a phosphodiester bond that links the 5’ and 3’ ends which have different polarities

Purines vs. Pyrimidines?

Purines: bicyclical rings → Adenine and Guanine

Pyrimidines: monocyclic rings → Cytosine, Thymine (DNA), Uracil (RNA)

What holds together the base pairs on a DNA double helix?

Complimentary base pairs are held together by hydrogen bonds where purines are bonded to pyrimidines

Major vs. Minor grooves on the DNA double helix?

Major groove → More exposed section of the DNA double helix, contains more “chemical information” that is more accessible to proteins

Minor groove → Less exposed section of the DNA that is not as accesible to proteins

How is the double stranded DNA split up for use during DNA replication?

One of the strands will be used as a “template strand”, the newly synthesized DNA strand will be complimentary ot this template strand. The other strand, that is unused, is referred to as the “coding strand” and will contain the same nucleotide sequence as the newly synthesized DNA strand. 

Describe DNA replication during Interphase? 

Chromosomes are duplicated inside the nuclear envelope

Describe DNA replication during Mitosis?

Nuclear envelope dissolves and mitotic spindle fibers attached to the centromeres of chromosomes, pulling apart the duplicated chromosomes. The cell then splits off and divides into two daughter cells and returns to interphase.

Three DNA sequence elements necessary to begin DNA replication? 

Telomeres → mark the ends of the cell, contain repeated nucleotide sequences that prevent valuable genetic information from being lost during replication

Replication Origins → ORIs are the point at which the DNA is seperated the replication beings

Centromeres → holds the strands together before the chromosome is ready for replication, also where the mitotic spindle fibers attached to seperate the duplicated chromosomes 

Explain why interphase chromosomes are more compact than mitotic chromosomes? 

When in interphase, the cell needs access to the genetic information located inside chromosomes to be able to adequately undergo DNA replication. During mitosis, the cell is dividing so the DNA inside the chromosomes is heavily compacted in order to maintain all the important genetic code stored inside the chromosomes 

Describe the chromatin & nucleosome relationship?

Chromatin is composed of many compacted nucleosome units. Nucleosomes are the basic unit that make up Eukaryotic chromosomes. Nucleosomes are composed of DNA tightly wound around histone octamer proteins. 

Briefly describe the levels of DNA packing?

The short region of a double helix DNA strand is tightly wound around histone octamers. These nucleosome core units are combine together to create a nucleosome which are tightly packed together to create a chromatin fiber. Chromatin fibers are folded into loops via nonhistone chromosomal “clamp” proteins. These folded loops compose an entire chromosome.

Describe histone octamer structure?

Histone octamers are composed of 2 of each of 4 different histone proteins: H2A, H2B, H3, & H4. The DNA double helix is tightly wound around this octamer but some of the histone proteins have tails that stick out of the octamer. 

Role of chromatin remodeling complexes and histone modifying enzymes? 

Chromatin remodeling complexes can adjust how the DNA is wrapped around a histone, making it more or less accessible as seen fit. Histone modifying enzymes take advantage of the histone protein tails that stick out of the octamer. These enzymes are able to add/remove acetyl, methyl, and phosphate groups to the histone tails which can alter chromatin structure. Both of these types of changes can alter chromatin to be more condensed (heterochromatin) or less condensed (euchromatin). 

What are Barr Bodies?

Barr Bodies refer to the inactivated sex chromosome in mammalian females. This most likely undergoes extensive histone modification that compacts it from Euchromatin to heterochromatin where it is no longer expressed and is “silenced”. 

How does DNA synthesis begin?

Initiator proteins bind to the replication origins on the original strand of the DNA. Hydrogen bonds between base pairs are broken and the exposed nucleotides are used as templates. Two replication forks are eat replication origin, they move in opposite directions which is why DNA replication is considered bidirectional. 

DNA polymerase

DNA polymerase can only add nucleotides to the 3’ -OH end of the growing DNA strand. For this reason, DNA is always said to be synthesized in the 5’ → 3’ direction and can only be synthesized this way. For this reason, the two newly synthesized strands are referred to as the leading strand, the continuous strand which is synthesized towards the replication fork movement, and the lagging strand, the discontinuous strand that contains Okazaki fragments and is synthesized away from the replication fork movement.

Describe DNA polymerase’s self correcting mechanisms?

DNA polymerase first proofreads previous nucleotide additions to check for incorrect nucleotide additions. 

RNA primers and Primase?

DNA polymerase can’t just begin synthesizing DNA; an enzyme known as primase synthesizes short RNA primers which give the DNA polymerase an area to attach and begin DNA synthesis. These primers are later removed by the DNA repair polymerase and nicks are sealed up by the DNA ligase which creates phosphodiester bonds. 

DNA helicase and topoisomerase?

DNA helicase is the enzyme that is responsible for unwinding the double stranded DNA helix. Topoisomerases relive the tension that builds up from torsional strain infront of a replication fork. It does this by creating a single strand break at the area of tension, the DNA can then freely rotate around the phosphodiester bond opposite of that single strand. This break is later sealed by the same topoisomerase. 

Lagging strand shortening in replication, how is it dealt with?

Telomeres and telomerase. The last RNA primer at the end of the lagging strand is removed and telomerase now comes in to extend the lagging strand with a telomere repeat sequence (full of nonessential genes). This telomere repeat sequence is complimented on the newly synthesized lagging strand by DNA polymerase. 

Describe depurination and deamination?

These are two chemical reactions that can seriously damage cell DNA. Depurination refers to the removal of a purine (adenine or guanine) from DNA. Deamination typically comes in one major type where cytosine is replaced with uracil in RNA. These two modifications can cause mutations that will be passed down due to nucleotide substitutions/losses.

DNA repair process for single strand breaks (3 major steps)?

1. Nucleases cleave off the damaged segment of DNA

2. DNA polymerase comes through and replaces the damaged segment of DNA with the correct nucleotides by using the other strand as a template.

3. DNA ligase seals the nics between the helix by reestablishing phosphodiester bonds.

DNA repair methods for double strand breaks? 

• Non homologous end joining: nucleases capture the drifting, broken DNA fragments and bring them back to the missing area where they are joined together by ligase. Results in some missing nucleotides.

• Homologous Recombination: Can only occur for a short period of time after DNA replication and before the cell divides into 2 daughter cells. In this case, the undamaged double strand will be used as a template to repair the double strand break. 

Major difference between the pentose sugar’s for RNA and DNA? Major difference between the base’s for RNA and DNA?

• Ribose has an extra hydroxyl (-OH) group that deoxyribose does not have. 

• Thymine in DNA contains an extra methyl (-CH3) group that uracil does not

RNA structure?

Typically single stranded but can have certain regions of temporary double strandedness with conventional and non-conventional base pairs. 

RNA polymerase

Unwinds the DNA helicase and uses one of the strands to attach ribonucleoside triphosphates to the growing single strand of RNA that is complimentary to the template strand of DNA. 

Promoters and terminators?

Promoters are necessary for RNA polymerase to begin RNA synthesis. The promoter region has a segment of DNA which the sigma factor (type of transcription factor) recognizes and allows the RNA polymerase to bind to. The terminator region, similarly, contains the segment of DNA that signals the RNA polymerase to detach. 

Where does mRNA translation occur?

mRNA is translated in the cytosol, it exits the nucleus via. nuclear pores 

In Eukaryotic cells, what does RNA processing compose of?

RNA processing into mRNA involves capping of the 5’ end, polyadenylation the addition of a poly-A tail at the 3’ end ,and RNA splicing in which the introns (noncoding, regions) are removed to bring the exons, coding regions, together and prepare for translation.

RNA splicing?

Specific intron sequences in pre-mRNA will indicate to the splicesome to remove these introns. This will bring together the exons and create mRNA which contains the coding sequences for the protein. The removed intron branch is referred to as a lariat and is released to be degraded in the nucleus. 

mRNA after processing?

Useful mRNA’s are moved from the nucleus to the cytosol where they are translated and then degraded to have their nucleotides reused. 

Decoding of mRNA into protein? 

mRNA is decoded in sets of 3 nucleotides which can be separated into different reading frames based on the start codon AUG. Ribosomes, composed of 4 rRNA molecules and 80+ ribosomal proteins, bind to the mRNA and contain 3 sites which tRNAs can enter: A, P, E sites. Decoding begins with a tRNA entering the P site and using its anticodon region to bind to the complimentary mRNA start codon. Another tRNA then enters the A site and the amino acid from this tRNA is attached to the growing peptide chain on the tRNA in the P site. Then the large subunit of the ribosome shifts, moving the used tRNA to the E site and the tRNA with the expanding peptide chain to the P site. The tRNA from the E site is ejected and another tRNA enters the A site to continue the process. This continues until a release factor binds with a stop codon binds to the A site and signals the release of the polypeptide chain (protein), the ribosome then dissociates as well.

Role of polyubiquitin markers?

Misfolded/damaged proteins can be marked by polyubiquitin chains which proteasomes recognize and use to know which proteins to degrade. 

Role of Homeodomain?

The homeodomain is a transcription regulator that consists of 3 α-helices. It works by examining the base pairs on the grooves of DNA to decide which genes to express.

Operon, operator, promoter?

A cluster of genes that are controlled by a single promoter are called an operon. A promoter is a sequence of DNA that the RNA polymerase can bind to to initiate transcription. The operator is located near/inside the promoter sequence and it acts as the “on/off” switch. 

Role of repressor proteins?

Repressor proteins tend to dimerize at the operator to prevent the RNA polymerase from accessing up/down stream promoter sequences or beginning transcription. 

Role of activator proteins?

Activator proteins bind to the regulatory sequences of DNA and help the RNA polymerase begin transcription by helping bind the RNA polymerase to promoters. 

Purpose of looped chromosome domain?

Looped chromosome domains are ideal for structural packaging of the genetic material. Additionally these looped domains help establish adequate positioning for genes and transcriptional factors. 

Describe the significance of the Pax 6 gene?

Pax 6 is a highly conservative master gene that is thought to control eye development in animals. The conservation of this gene suggests that animal eyes evolved from a common ancestral prototype. 

Directing the formation of different cell types in culture?

Specific transcriptional factors can be used to reprogram differentiated cells into pluripotent stem cells which can differentiate into any type of cells in your body. 

Positive feedback loops in differentiated cells? 

A temporary signal in the parent cell is used to turn on gene A. Protein A is used to keep gene A on; a positive feedback loop has been created. This memory is passed onto all daughter cells; regardless of whether or not that temporary signal is there, daughter cells will keep gene A on using protein A

Role of methyltransferase + histone modifying enzymes in replicating methylation?

Methyltransferase ensures that the newly synthesized DNA strands will methylate the necessary base pairs to match the methylated base pairs on the template strand. This pattern holds true for methylated histones as well, where histone modifying enzymes will establish the same histone modification pattern as the template.

Role of miRNA?

miRNA’s guide RISC complexs to complimentary mRNA’s, either degrading it to use it’s nucleotides later or simply just repressing the mRNA 

Role of siRNA in RNAi? 

Foreign double stranded RNA is cleaved by the dicer enzyme, remaining pieces are referred to as siRNA’s. RISC proteins work to degrade on strand of these siRNA’s and use the other strand to detect complimentary foreign RNA’s and silence them. 

List the mechanisms by which genes and genomes can be altered?

• Gene mutations

  • Mistakes in the recipe

• Regulatory DNA mutations

  • Changes to the on/off switches

• Gene duplication and divergence

  • Use the same recipe with different spices sprinkled in

• Exon shuffling

  • Mix & match lego pieces of the proteins

• Transportion 

  • Mobile DNA jumps around

• Horizontal Gene Transfer 

  • DNA from your neighbors is borrowed 

  • DNA transfer that is not done from parent to offspring

Germline vs. Somatic Cell functions?

Germline cells are essentially the genetic heritage that is passed off to the next generation. These cells give rise to gametes (egg/sperm cells) which separately contain one chromosome but can combine to form a zygote with the right number of chromosomes. Mutations in germline cells will be passed onto and expressed in the next generation. Somatic cells are the “body builders” and make up all the other cells of your body. The genetic information from these cells is not passed onto the next generation. 

Role of homeobox genes?

Master regulatory genes that control the body’s plan for developing the embryo by specifying the parts of the body.

DNA only transposons and their movement mechanisms?

DNA only transposons can move to new chromosomal locations. They can move by cut and paste transposition where an element is cut out of the donor DNA and moved to the target DNA. They can also move by replicative transposition where the mobile genetic element is copied by DNA replication.

Retrotransposons and their movement mechanisms?

Similar to the transposons, these move via copy and paste transposition but by using an RNA intermediate. Here the donor segment is transcribed into RNA and then back into copied, double straded DNA where it is then inserted into the target DNA.

How do viruses replicate?

They utilize the host’s machinery to reproduce. They enter the cell and then release their DNA into the cell where it is replicated, transcribed, and translated into protein that then assembles into other virus particles which will exit the cell. 

Retroviruses and their role?

Retroviruses enter the host cell with an RNA genome. They utilize reverse transcriptase to convert RNA into an RNA/DNA hybrid and then into double stranded DNA. This double stranded DNA is then integrated into the host’s chromosome and becomes a permanent part of the host’s genome.