BIOL 112 Final Exam

B

explain the steps of bacterial cells translation (electron micrograph)

1. First ribosome binds to the mRNA

2. A second ribosome binds after first ribosome has moved downstream, polypeptide chain is being synthesized by ribosome 1

3. a third ribosome binds as 1 and 2 are moving along the mRNA downstream

what does CRISPR stand for

Clustered Regularly Interspaced Short Palindromic Repeats

what is the CRISPR/Cas system

• a widespread class of immunity system that protect bacteria and archaea against phages and other mobile genetic elements

• use repeat/spacer-derived short crRNAs to silence foreign nucleic acids in a sequence specific manner

what are CRISPR arrays

• contain all of the viral DNA samples together in a single repeated array

• a storage bank that records previous encounters with viruses that infect the bacterial cell

spacers vs repeats

• the CRISPR array itself is a permanent part of the bacterial genome and can therefore be passed to offspring cells

• each spacer sequence is taken from a different virus

• different spacers = different viruses

what is crRNA

• the RNA that bacterial cells transcribe from the CRISPR RNA

• crRNA is cut into smaller sequences that contain spacer and repeat sequences

what do Cas9 enzymes do

• use crRNA to target viral DNA

• crRNA binds to Cas9 enzymes

CRISPR-Cas9 complex

• multiple copies of Cas9 proteins each with a piece of crRNA

bottom strand

how does bacteria defend itself from new viral infections

1. finding viral sequences

   • a new virus infects the cell and inserts the viral DNA into the genome of the bacteria

   • the CRISPR-Cas9 complex scans the genome of the bacteria to find viral sequences

   • they use the crRNA as a guide to find a sequences match to the virus genome

2. matching crRNA with DNA in bacteria

   • the cas9 opens opens the sequence of viral DNA and checks it against the crRNA samples it is carrying

   • if it matches crRNA-Cas9 and the genome forms complementary base pairing

   • the crRNA and DNA match are locked in

3. cut out viral DNA sequence

   • when locked in, the CRISPR-Cas9 will proceed to cut out the sequence that matches the spacer (i.e. original viral DNA sequence)

   • Cas9 cuts up viral DNA

how does cutting/editing defend against viruses

• cut in the middle of an important viral gene, killing the virus

• other cas9 enzymes maybe carrying crRNA that match other parts of the viral DNA, thus increasing the chances of cutting up and killing the viral DNA before it can hijack the cell

what is gRNA

• guide RNA

• cut any DNA of interest and then edit it

• this guide RNA sequence hybridizes to the top strand of this DNA (B sequence)

• C sequence hybridizes to the bottom strand of this DNA

what happens when the DNA is cut

• the cell’s DNA repair system are turned on

• scientists can then introduce a customized DNA template for the repair system to use, changes DNA sequence

what does gene regulation mean

• a cell does not express all of its genes all of the time, very selective about the genes they express

  • how strongly they are expressed (high vs low)

  • when they are expressed

• a gene is said to be expressed when the gene product is actively being synthesized and used in a cell

what is a constitutively expressed gene

• a gene that is expressed all the time because its gene product is needed all the time

  • eg. rRNA, tRNA, RNA pol, ribosomal proteins, amino acyl tRNA synthetases

how does promoter strength affect gene expression

• determines how frequently transcription is initiated

  • this determines how many RNA molecules are made

how does mRNA half-life affect affect gene expression

• how quickly the mRNA is degraded after it is made

• the longer mRNA lasts, the more protein copies can be translated per unit time

• only applies to protein encoding genes

• tRNA and rRNA are stable RNA so not degraded

transcriptional level of control examples

1. promoter strength - how strongly RNA pol + transcription factors bind to the promoter

2. operons - regulatory proteins binding to operator

what is an operon

• share a promoter and termination sequence but have multiple coding regions

• one mRNA is produced yet multiple proteins are translated

what can be found in operons

• a regulatory region called the operator can be found either upstream or downstream (and sometimes overlapping) the promoter

• the regulatory protein binds to an operator region

basal definition

• means base, minimal

positive regulation

• regulatory protein binds a region by the promoter and increases transcription

• regulatory protein called an “activator” protein

  • eg. MalT

negative regulation

• regulatory protein binds to a region by the promoter and decreases transcription

• regulatory protein called a “repressor” protein

  • eg. LacI

catabolic operons

• lac and mal operons are examples

• some cells use lactose or maltose as a source of energy and carbon

• the cells only needs to synthesize the proteins in the operons if the sugars are present in the environment

• the proteins produced in these operons function to break down sugars

what are inducers

• when present, the lac and mal operons are expressed at high levels

what is a co-repressor

• if the presence of a signal molecule results in low levels (to zero) of operon expression, this is often referred to as a “co-repressor”

anabolic operons

• produce proteins that are involved in the synthesis of the signal molecule

signal molecule (eg. arginine)

• the operon are expressed in the absence of the signal molecule, the signal molecule is a co-repressor

• i.e. helps the repressor protein, repress the expression of the operon

function of DNA replication

• DNA encodes all of the proteins and RNA in the cell to allow for proper cell function

• new cells will need a copy of the genome

key properties of DNA polymerase

• DNA pol reads the sequence on a template and links nucleotides together, i.e. reads the template 3’ to 5’ and synthesizes nes DNA in the direction 5’ to 3’

• DNA pol cannot start on its own, must always start from an existing 3’OH end of an existing template

• in the cell, the primer is RNA

what are requirements for polymerization of RNA/DNA

1. requires an enzyme (DNA or RNA polymerase)

2. requires a form of energy

   • this energy form is a monomer with 2 or more phosphates, most common forms = triphosphates

polymerization of DNA or RNA

• the 5’ carbon of a new monomer is added to the 3’ carbon of the existing strand (or 1st monomer)

why are monomers shown as a triphosphate?

• binding of three phosphates creates an unfavourable state (3 negative ions in close proximity)

• removing the outermost phosphates release energy

• the energy can be used to link the monomer to the polymer

explain why DNA replication is semi-conservative

• each cell will receive an original template strand that is base paired with a newly synthesized strand

what is the rate of replication

• ~1000 base pairs/second

how does linear DNA replicate

• needs more than one origin of replication (OriR)

how does circular DNA replicate

• bacterial cells can replicate in a circle (both directions), ends will meet up

• usually has 1 origin of replication (OriC)

what are three problems the cell has to solve to replicate DNA

1. separate the DNA strands only a little at a time, can’t separate an entire genome

2. make primers for DNA polymerase

   • can’t make a DNA primer without a DNA primer

   • cell makes an RNA primer - can start without a 3’ OH

3. allow synthesis to happen simultaneously from the two template strands

   • DNA strands are anti-parallel, yet DNA polymerization has to occur in the 5’-3’ direction

   • synthesis has to happen in opposite directions

solutions to the problems (DNA replication)

1. helicase unwinds DNA at replication fork

2. RNA primase → RNA polymerase (RNA primase) makes RNA primers, DNA pol can add dNTPs to the 3’ OH end of this short RNA sequence

3. from the replication fork - synthesis occurs in the opposite directions (i. e. in one direction on one strand and in the other direction on the other strand)

   • but overall, replication must progress on both strands in the direction of the growing replication fork

what do primers do

• direct the start of polymerization

• DNA pol cannot start on its own, must always start from an existing 3’ OH end of an existing template. in the cell, the primer is RNA

• primase is an enzyme that creates the primer

A

what happens as the replication fork opens?

• at first, both strands (leading and lagging) are synthesized as the replication fork

• the replication fork has opened further to the left so need to start a new Okazaki fragment

  • as replication fork opens further, the top strand has DNA pol moving to the right and leaving a gap at the fork, so another Okazaki fragment is added to join the first fragment

discontinuous vs continuous synthesis

• leading strands are referred to as continuous synthesis

• lagging strands are referred to as discontinuous synthesis

DNA polymerase I function

• removes the RNA primer (red) and replaces it with DNA

DNA ligase function

• joins all the fragments together

• joins all Okazaki fragments

• joins leading and lagging strands at origin of replication (pink circles)

topoisomerase function

• relieves stress on winding helix

single-stranded binding proteins

• keep the single stranded DNA apart

metabolism definition

• the set of biochemical reactions that transforms biomolecules and transfers energy

catabolism

• energy released from “breaking down” or degrading molecules can be converted into useable forms of energy for the cell

• note: ATP synthesis is coupled to substrate catalysis

anabolism

• “making” or building molecules and structures in the cell uses energy

• note: ATP catalysis is coupled to substrate anabolism

what are nutrients

• substances taken from the environment by organisms for their growth, development and to sustain them

what is a bio element

• elements found in cells and that are required for cellular function

autotrophs

• organisms that can convert CO2 into organic carbon

heterotrophs

• organisms that get organic carbon from other organisms

organic compounds

• glucose (C6H12O2)

• glycerol

inorganic compounds

• water

• nitrite

• hydrogen sulfide

• ammonia

what is cellular respiration

• oxidation of glucose to carbon dioxide

• glucose donates electrons to O2

• the carbons in glucose end up as the carbons in CO2

• 4 stages

  • 1. glycolysis

  • 2. pyruvate oxidatiaon

  • 3. citric acid cycle

  • 4. oxidative phosphorylation

location of cellular respiration in eukaryotes

• step 1 = cytoplasm (glycolysis)

• step 2 + 3 = mitochondria (pyruvate processing + citric acid cycle)

• step 4 = mitochondria (oxidative phosphorylation)

location of cellular respiration in bacteria

• steps 1-3 = cytoplasm/cytosol (glycolysis + pyruvate processing + citric acid cycle)

• step 4 = oxidative phosphorylation (cell membrane)

what are some high energy intermediates

• ATP, NADH, FADH2

• these are usable forms of energy for the cell

where does a cell get the “energy” from

• from nutrients

• energy is captured by oxidation and reduction reactions

• nutrients are electron donors

• the electron acceptor molecules capture some of the energy loss from the electron donors

oxidation

• loss of electrons by an atom

• and usually gains O atoms or loses H

reduction

• gain of electrons by an atom

• usually also gains H atoms or loses O

A

what is fermentation

• a metabolic process that converts sugars to acids, gases or alcohols

• fermentation is an alternate pathway after stage 1, not all cells can ferment

• no terminal electron acceptor

• if glucose is available, the cell will undergo glycolysis and produce ATP and NADH but ends there

respiration vs fermentation

• respiration: ATP made in stage 1, 3, 4 i.e. cell makes most of its ATP by oxidative phosphorylation

  • all 6 carbon atoms in glucose are completely oxidized to CO2

  • oxidized C atoms discarded as CO2 waste

  • more ATP can be synthesized if the cell can complete all 4 stages of cellular respiration

• fermentation: cell makes all of its ATP by substrate level phosphorylation (in glycolysis)

  • no net oxidation of C

  • electrons removed from some C atoms and returned to others

  • the products of fermentation are waste products

  • much less ATP if a cell can only complete stage 1 (glycolysis) and fermentation

when cells respire, what element do they use as a terminal electron acceptor

O2

why can’t pyruvate continue in cellular respiration

• most likely reason is lack of terminal electron acceptor O2

what are the two types of fermentation

• ethanol fermentation

• lactic acid fermentation

can pyruvate undergo fermentation?

• if the cell has the capacity to do so

C

slp = substrate level phosphorylation

A

fermentation if there is no terminal electron acceptor

• pyruvate will undergo fermentation

• NADH will deliver electrons to fermentation intermediates

what is being described in this picture

• NADH donates electrons to re-generate NAD+

• Now, NAD+ can return to glycolysis to pick up more electrons

B

D (lactic acid)

E

D

E

C

B

glycolysis

• glucose is broken down to pyruvate

• ATP and other high energy intermediates are produced

• occurs in cytosol

• goal is to degrade glucose, extract energy from the reduced form of carbon (glucose) in several steps, synthesize high ATP and NADH (electron carrier), create pyruvate - a molecule that can be used in different pathways

• uses 2 ATP and synthesizes 4 ATP

• synthesizes 2 NADH (4 electrons - 2 to each NAD+)

explain phase 1 of glycolysis

• energy investment phase

  • 2 ATP used to phosphorylate a 6C sugar with 2 negatively charged phosphate groups

explain glycolysis phase II

• energy payoff phase

  • 4 ATP made by substrate level phosphorylation

  • 4 electrons removed (from G3P) to reduce 2 NAD+ to 2NADH

oxidized form of NAD

• NAD+

  • can take up/accept 2 electrons

reduced form of NAD

• NADH

• can donate 2 electrons

what is substrate level phosphorylation

• enzyme catalyzes transfer of phosphate from a phosphorylated molecule to ADP

location of substrate level phosphorylation

• euk cytosol or mitochondrial matrix

• cytosol for bacteria

SLP vs oxphos

• SLP involves enzymes and substrates that phosphorylate ATP

• oxphos involves membrane-bound enzymes and H+ gradient drives ATP phosphorylation

C

D