modern bio unit 2

What is the Gibbs Free Energy equation?

∆G = ∆H - T∆S

What does the ∆G mean?

free energy change

What does the ∆H mean?

enthalpy change (products more stable -∆H, products less stable +∆H)

Which is more stable OH or CH? Which has more PE?

CH has more PE but less stable

What is the T∆S?

temperature (kelvin) and change in entropy

∆G < 0 is what reaction?

Spontaneous and exergonic, products are more energetically favorable than reactants

∆G > 0 is what reaction?

Not spontaneous and endergonic, products are less energetically favorable than reactants

Why don’t most chemical reactions happen normally?

Because of the high activation energy need to put it through transition state

What do enzymes do?

Lower activation energy of reactions, protein RNA catalysts

Describe how enzymes facilitate reactions

Reactants bind to active site of enzyme, interactions between enzymes and substrate lowers activation energy, products go to changed state

Explain how Exergonic reactions can be coupled with endergonic reactions

Exergonic reactions with ATP charge substrates to make endergonic reactions energetically favorable (spontaneous)

How do competitive inhibitors work?

Competitive inhibitors take over the active site and block the substrates from bonding, these bind reversibly through noncovalent interactions

How do Allosteric inhibitors work?

Allosteric inhibition happens when the ligand change the enzyme shape and thus change the active site to either allow or restrict substrate bonding

What are metabolic pathways?

Pathway where enzymes convert substrates along multiple enzymes to get a final product

How does feedback inhibition work?

The product from an enzyme or pathway of enzymes inhibits earlier enzymes in the pathway. Allosteric binding

In Glycolysis how is ATP formed?

Substrate level phosphorylation is an enzyme catalyzed reaction that results in ATP production

Explain redox reactions

Reduction Oxidation, LEO GER, lose electron oxidation, gain electron reduction, ex) NAD+ to NADH is a reduction

Maximum ATP produced in cell resp

29 total, 4 in glycolysis 25 in mitochondria

What is the makeup of nucleotides

Phosphate group on 5’ carbon of 5-carbon sugar along with nitrogenous base on 1’ carbon

What are the key differences between DNA and RNA

RNA uses ribonucleotides (hydroxyl group on 2’carbon) DNA uses deoxyribonucleotides (H on 2’carbon). RNA uses Uracil (U) DNA uses Thymine.

How do nucleotides bond with other nucleotides?

condensation reactions to form phosphodiester bonds, between 3’ carbon of one and hydroxyl on phosphate group

What is nucleotide sequence directionality?

5’ end (unlinked phosphate) to 3’ end (unlinked hydroxyl), new nucleotide can only be added to free 3’ -OH

Polymerization of nucleic acids requires an energy source. Why?

This is because products of reaction are less stable than the reactants, thus a nonspontaneous, endergonic reaction

How is base pairing in DNA formed?

By hydrogen bonding between purine-pyrimidine pairs. Adenine - Thymine and Guanine - Cytosine

What is RNA?

Less stable, more reactive than DNA, usually single-stranded, but can bond together to form 3d structure, first cell thought to have RNA based genome

Roles of RNA

information messenger, information translator, enzyme, etc

Why does replication start at multiple points in eukaryotic cells?

Because the genome for eukaryotes is longer and its more efficient to start multiple times and have them fuse to make a larger bubble

Semi-conservative replication, what does that mean?

The parental double strand is split in two, then each is combined with a daughter strand, thus each strand is semi-conserved.

What is Helicase?

Enzyme that breaks hydrogen bonds holding two template strands together using ATP.

What provides energy for DNA replication?

The potential energy of ATP

Why is single stranded DNA-binding proteins needed?

Single stranded DNA is prone to join back together due to the attraction of base pairs causing it to be favorable to stick back together

What is Topoisomerase

Enzyme that untwists the DNA by breaking phosphodiester bonds and rejoins bonds once helicase catches up.

What is Primase?

Enzyme that catalyzes the synthesis of RNA primers that are required

Why is RNA used as a primer?

DNA primase needs a free 3’-OH but RNA does not.

What is DNA polymerase III?

An enzyme that catalyzes the addition of nucleotides beginning from a free 3’-OH group, only works in 5’ to 3’ direction

What are leading vs lagging strands?

Leading strand, DNA synthesized from the 3' end of the original, does not need to stop. Lagging goes backwards from the DNA unraveling and thus has Okazaki fragments

What are Okazaki fragments?

Fragments of lagging strand that results from DNA being made in the opposite direction

What does DNA ligase do?

Joins the okazaki fragments into a continuous strand

Describe the process of lagging strands

Primer added, first fragment synthesized by DNA Pol III goes till next primer making fragment, primer replaced by DNA pol, gap closed by DNA ligase

What happens at the end of DNA replication?

The leading strand continues all the way but the leggings strand is cut off, as after the primase is taken out there is no synthesis after that, thus the end is unreplicated.

What are Telomeres?

expendable stretches of DNA at the ends of chromosomes, as a cell’s telomeres shorten over rounds of DNA replication, signals are sent to stop the cell from dividing

What is Telomerase?

protein-RNA enzyme that generates telomeres at the ends of chromosomes

Where is Telomerase only active?

In cells that divide rapidly (germ cells and stem cells)

Describe how Telomerase extends DNA

In the lagging strand, the DNA is left unreplicated. The Telomerase extends that end with its own RNA template, shifting and repeating activity, and the extended single stranded DNA acts as a template

What determines the sequence of telomeres and why are they repeating?

The RNA template in the Telomerase. It is repeating to continue extending off of the same template

Difference of mutations in somatic vs germ cells

Mutations in somatic cells result in clones of the mutant cells but will not be inherited by offspring, while in germ cells the mutations may be passed down

Sources of mutations

Mistakes during DNA replication, chemical instabilities in DNA, damage by sunlight (UV), x-rays, chemicals

What keeps mutation rates in check?

proofreading by DNA Polymerase, mismatch repair, nucleotide excision repair

Describe proofreading by DNA polymerase

when DNA pol makes a mistakes, it pauses synthesis, uses its exonuclease activity to remove the incorrectly paired nucleotide, then resumes

What is mismatch repair

Where most mistakes can still be caught. Uses enzymes to correct mistakes during replication that were missed by proofreading

Describe how mismatch repair works

distorted DNA detected by repair proteins, Endonuclease breaks portion of backbone of daughter strand, repair exonuclease removes nucleotides, DNA polymerase closes gap and ligase seals backbone

How does UV light affect DNA?

Light can produce enough energy to consecutive thymines to make them bind to each other. Thus making them not free to make base pairs with a complementary strand

How does chemical instability affect DNA?

For example, deamination of Cytosine in DNA changes it into Uracil. This is spontaneous.

What is nucleotide excision repair?

Repair of damaged or irregular nucleotide pairs after replication. Enzyme cuts DNA, helicase unwinds and removes region with damaged bases, polymerase fills gap, and ligase links newly synthesized dna

What is XP and how does it affect cells?

XP is a mutation that causes cells to be more vulnerable to UV damage and unable to repair that damage, thus making sunlight very dangerous.

What are potential consequences if DNA damage is not repaired?

Permanent mutation, loss of function, cancer

What is mRNA?

The information carrier that transcribes from DNA to translate that information and make protein at ribosomes

What is transcription and translation?

Transcription is copying information in the same language, DNA to RNA, translation is converting one to another, mRNA to amino acids

What is RNA polymerase?

synthesizes RNA in the same direction as opening DNA template. The produced sequence will be complementary to the template strand and identical to the nontemplate strand. RNA polymerase does not need an RNA primer

Qualities of genetic code

it is redundant, unambiguous, nearly universal, conservative

What are the different consequences of point mutations on proteins?

Silent, missense, nonsense, frameshift. Point mutation is one nucleotide change to DNA sequence

What are silent mutations

mutation that does not alter amino acid sequence. Changes mRNA sequence but not phenotype amino acid.

In which base position of a codon are we most likely to see silent mutations?

3rd position

What are missense mutations?

Change of one amino acid. Can or cannot affect protein function. Sickle cell anemia is a result of this

What are nonsense mutations?

Introduction of an early stop codon. Almost always negatively affects protein function.

What are frameshift mutations?

Addition or subtraction of base that causes all the codons after the mutation to shift, thus ruining protein function.

How does RNA polymerase synthesize RNA?

RNA polymerase unwinds DNA on its own, then uses one template strand and transcribes gene to make a RNA transcript

What are promoters and terminators?

DNA sequences where RNA polymerase will initially bind at the start of genes, and end at terminator sequence, synthesized in the 5’ to 3’ direction

How does RNA pol in prokaryotes find the gene?

Through sigma factors that lead the RNA polymerase to the gene, then bind to the promoters along with the RNA polymerase

Describe prokaryotic transcription

Initiation — The sigma factor guides RNA polymerase to the promoter, recognizing the –35 and –10 boxes, which orient the polymerase in the correct direction. RNA pol then unwinds the DNA (no primer needed — RNA pol starts synthesis on its own).

Elongation — Sigma factor is released. RNA polymerase synthesizes RNA 5'→3', reading the template strand 3'→5', adding ribonucleotides to the 3'-OH. The transcript is complementary to the template strand and identical to the coding strand (with U replacing T).

Termination — The polymerase transcribes a termination sequence that causes the RNA to fold into a hairpin loop. This destabilizes the RNA–DNA pairing, the transcript is released, and transcription ends.

What are transcription factors?

Factors in eukaryotes that recognize specific DNA sequences in promoters and other regulatory sequences

Describe splicing in Eukaryotes

Newly transcribed RNA are nonfunctional and longer than they need to be, thus the introns must be spliced out and the exons need to be kept

Alternative splicing

Another way to regulate protein expression, exons can be removed and result in different resulting gene expressed.

Parts of mRNA in Eukaryotes

5’ cap, poly A tail, that stabilize RNA against degradation

What are the main parts of ribosomes?

Large (50S) and small (30S) subunits . The small subunit holds mRNA in place and the large subunit forms peptide bonds. Ribosomes are made of rRNA and many proteins

What is tRNA?

Transfer RNA that act as adaptors for mrRNA to amino acids, contains CCA sequence where amino acids are initially covalently bonded

Describe wobble base pairing

Where the third base in the codon doesn’t always pair with the anticodon base as the amino acid can be recognized with the first two bases anyways

How does tRNA bond the amino acids

They need to be charged with high energy, each enzyme binds to one uncharged tRNA and the amino acid, thus using ATP to make a high energy covalent bond between the tRNA and amino acid.

What are the phases of translation

initiation, elongation and termination

Initiation in translation

mRNA binds to small subunit that tries to find start codon, then once Met-tRNA in P site, finds start codon large subunit of ribosome binds

Elongation in translation

second tRNA binds to A site, then tRNA and amino acid bond breaks and forms peptide bond, then tRNA moves down a spot. Then repeats from A site (come in) P site (bond) and E site (exit)

Termination in translation

release factor binding to A site does not create peptide bond, thus tRNA and polypeptides leaves and large subunit separates, ending translation

Protein release factor must have a similar shape to what so it binds to A site?

Similar shape to tRNA

Goal after translation

protein is folded, folding begins during translation, as soon as N-terminus emerges from ribosome

What is PCR?

PCR (polymerase chain reaction) is used to amplify a specific DNA region of interest

How does PCR work?

1. Creates reaction mixture of dNTPs (building blocks) and primers along with Taq polymerase (important bc heat stable), 2. Denature with heat, 3. Primer annealing DNA synthesis needs primer to start, 4. Extension in 5’ to 3’ direction. This process is exponential

Agarose gel electrophoresis

process that pulls dna fragments using electric field to pull fragments towards positive end, smaller move faster and more and larger move slowly

What is Sanger sequencing?

Process that uses many rounds of PCR on a sample to be sequenced using a mix of dNTPs and color labeled ddNTP that terminate a reaction when incorporated into the product. Thus color can indicate base identity.