Bio 161 exam #3 content

what ar the key concepts of gene expression regulation?

• not all genes in a cell are expressed (transcribed into RNA and translated into protein) at any given time. Some are “on“ and some are “off“

• in multicellular organisms, all the different cells in one individual have the same DNA but differ because they express different genes and thus make different proteins

• which particular genes are being expressed can change, due to internal conditions or in response to signals from the external environment

coordinate

turning on multiple genes with related functions at the same time, but do so in different ways

• done by both prokaryotic and eukaryotic organisms

regulating gene expression

• only a subset of organism’s genes are being expressed into products at any given time

• gene expression can be regulated at any step from the accessibility of DNA to transcription machinery to the creation and longevity of a functional protein product

what is the importance of how transcription is regulated?

the frequency at which a gene is transcribed is a major determinant of the amount of product made

• no binding= no transcription

• weak/infrequent binding= some transcription

• strong/ frequent binding= high transcription

• Not on or off, there’s an in between

where does RNA polymerase bind in prokaryotes?

binds directly to the promoter

how does RNA polymerase bind directly to the promoter?

• two short sepreate consensus sequences in the promoter are upstream from where transcription begins, RNA polymerase recognizes and bind these sequences

• the sigma subunit of RNA polymerase binds to specific promoter sequences (many different sigma subunits)

consensus sequences

the most frequent nucleotides found at each position when the similar sequences from various genes are aligned

• short stretches of highly similar DNA sequences that appear in many different genes

what does the consensus sequences being present in many promoter regions imply?

that these specific nucleotides are important for RNA polymerase protein to bind

• these nucleotides must be key to interacting with the proteins

DNA sequences-specific binding proteins

proteins bind to specific sequences (order of nucleotides) on DNA

• the r-groups of the protein’s amino acids can make bonds with the edges of the nucleotides

• different DNA-binding proteins bind different specific sequences

• In prokaryotes, one subunit of RNA polymerase is a DNA sequence-specific binding protein that binds directly to the promoter

how does eukaryotic transcription initiation differ from prokaryotic?

• eukaryotic and prokaryotic promoters have different consensus sequences (eukaryotes have a TATA box)

• eukaryotic RNA polymerase binds to promoter sequences indirectly: it cannot bind to promoter DNA on its own and instead binds proteins that bind the promoter

• a set of general transcription factor proteins first bind to a sequence with many As and Ts called TATA box, and then recruit RNA polymerase, and other general transcription factors to bind

transcription factors

proteins that bind DNA and control the rate of transcription. Most are DNA sequence-specific binding proteins

General transcription factors

proteins that help eukaryotic RNA polymerase bind to the promoter and initiate transcription

• eukaryotes

specific transcription factor

other transcription factor proteins that may bind to the promoter or elsewhere, to regulate the rate of transcription

• prokaryotes and eukaryotes

activator Transcription factors

increase gene expression

repressor transcription factors

decrease gene expression

activator protein

bind regulatory DNA sequences and stabilize increase binding strength of RNA polymerase, thus increasing gene expression

• in either case (general transcription factor or specific transcription factor), only a modest amount of transcription may occur if no activator protein is present

repressor proteins

bind (different) regulatory DNA sequences and reduce or block binding of RNA polymerase, thus decreasing gene expression

how do activators and repressors bind?

• each activator or repressor protein binds to its own particular DNA consensus sequence

• cells specific gene expression depends on the different combinations of transcriptional activators and repressors

Coordinate regulation

Refers to expressing a set of genes with related functions at the same time

1. Operons → prokaryotes

2. Specific transcription → prokaryotes and eukaryotes

Operon

Arrangement of multiple genes under the control of a single promoter. Leads to production of (multiple copies of) a single mRNA that encodes multiple proteins

• several genes under the control of 1 promoter

How do prokaryotes use coordinate regulation using operons?

• multiple genes controlled by one promoter

• Transcribes into a single mRNA

• Individual start and stop codons in the mRNA allow translation into separate proteins

• In prokaryotes, genes with related functions are frequently found in operants, this allows one stimulus to simultaneously lead to expression of several genes

How do eukaryotes use activator and repressor transcription factors in coordinate regulation?

• eukaryotic genomes sometimes but rarely have operons (comes back to recognition of specific sites)

• One stimulus can change the activity of a transcriptional activator or repressor, multiple separate genes in different chromosomal locations may then be regulated by copies of that transcription factor, if they have the binding site for it (done by both prokaryotes and eukaryotes but more by eukaryotes)

Coordinated gene regulation in prokaryotes:

Related genes are in an Operon, side-by-side under one promoter. Activator transcribes multiple genes at once

Coordinated gene regulation in eukaryotes

One activator protein binds in front of a set of multiple genes, which cab be on separate chromosomes, activating all of them

Why is DNA replicated?

• before a cell divides it copies its DNA

• Makes two double-stranded molecules each containing one original (parental) and one newly synthesized strand through a process of division called mitosis, each new cell will receive an identical copy of the parent cell’s genome

• 1 cell with 46 chromosomes → 2 cells with 46 chromosomes each

Origins of replication

DNA sites where DNA replication begins

Topoisomerase

Relieves over-winding

• relieves stress, untwists the over twisting

DNA Helicase

Breaks H-bonds (“unzips”)

• important to expose each strand as a template fro new strand synthesis

Single strand DNA binding proteins

Keep strands from coming back together

• prevents from reforming H-bonds

• Both strands in both directions from an origin of replication serve as templates from DNA replication

What is the difference between a single origin of replication in prokaryotic and eukaryotic cells?

• always start at the same spot

• In prokaryotic cells, origins are specific DNA sequences. Less clear what defines an origin of replication in eukaryotic cells

• B/c eukaryotic chromosomes are bigger and linear multiple origins, continue until they run into each other

How are DNA and RNA polymerase similar?

Both of these enzymes:

• “Read” a DNA template strand

• Build/synthesize in the 5’-3’ direction

How is DNA polymerase different than RNA polymerase?

• DNA polymerase needs help to begin the process of nucleotide polymerization. This help comes from a primer that provides a free 3’ OH

• DNA polymerase uses dNTPs → the A/C/G/T nucleotide building blocks (dATP, dCTP, dGTP, dTTP)

• once this short RNA primer is synthesized by primase, the DNA polymerase III will elongate the nucleic acid chain

Primer

A short nucleic acid sequence that acts as a starting point for DNA synthesis

• composed of RNA nucleotides

primase

enzyme that binds on RNA primer

• DNA synthesis starts with this

how does the replication bubble work?

• a replication bubble is composed of two replication forks

  • proteins that are involved in initiating replication will bind to an origin of replication, unwind the DNA and begin the process of replication

  • the replication is bidirectional

• each frok has a leading strand and a lagging strand

bidirectional

replication is ocuring on both strands and in bith directions at the same time

leading strand

continuous synthesis

lagging strand

discontinuous synthesis

what are okazaki fragments?

discontinuous pieces of DNA

• the lagging strand is composed of okazaki fragments

• each okazaki fragment requires its own RNA polymerase

• the replication is discontinuous because each fragment is independently synthesized

DNA polymerase III

extends from the primer to synthesized complementary DNA strand

sliding clamp

attach to DNA polymerase and keep it secured to the DNA strand

• allows DNA polymerase to be processive

processive

working quickly without relasing the template

what happens to the okazaki fragments at the end of DNA replication?

RNA primers are replaced with DNA and gaps are sealed by DNA polymerase I and RNA Ligase

DNA polymerase I

degrades the RNA primer and fills in with DNA

RNA Ligase

seals teh remaining gap by forming a phosphodiester bond

DNA replication vs. transcription of RNA similarities?

• a polymerase reads a DNA template from 3’-5’ to build a new neculic acid strand 5’-3’

differences if DNA replication compared to RNA transcription

• make exactly one copy of the full length of every chromosome

• both DNA strands in a double-stranded helix serve as templates, both are copied

• synthesis begins at origin of replication and ends with DNA polymerase runs into a double-stranded region

• DNA polymerase uses an RNA primer to begin synthesis; unable to start synthesis by creating a bind between the first two nucleotides

  • no termination sequence fro DNA poly. III

  • DNA poly is faster and makes fewer mistakes

differences of RNA transcription compared to DNA replication

• make many copies of small segments of a chromosome

• one of the DNA strands in a double-stranded helix serves as the template

• synthesis terminates at a specific sequence

• once RNA polymerase binds to the promoter region, it is ready to create a bond between the first two nucleotides

How does DNA polymerase make less mistakes?

• DNA polymerase has “proofreading” ability- it senses addition of the wrong nucleotide, cuts it out, resynthesizes using the correct nucleotide

  • a subunit of the enzymes preforms this proofreading

    • corrects most of its mistakes right away

• wrong nucleotide accidentally inserted → DNA poly. III backs up, cuts out wrong nucleotide → DNA pol. III moves forward again, adding correct nucleotide

mismatch repair

removes mismatched nucleotide

excision repair

removes chemically damaged nucleotide

what does the effect of a mutation depend on?

• whether the mutation is in a gene

• if it is in a gene, what part of the gene (promoter, UTR, protein-coding regions, introns)

• if in protein-coding sequence, then it depends on how it affects the codons(s)

what happens when a gene has a mutation, a change in the genetic code?

• creates genetic variation

• the following terms apply to mutations in protein-coding regions of genes

silent mutation

no effect on the protein structure

• no effect because of redundency in the genetic code

  • no difference in amino acid

missense mutation

changes one amino acid in the protein sequence

• one codon is changed causing a new amino acid

• the effect depends on the specific site in the protein and the specific amino acid substitution

nonsense mutation

introduces an early stop codon into the protein sequence

• changes a codon to a stop codon, truncating the protein

• usually result in “loss of function“ mutations since the shortened protein is usually non-functional

  • can have bad effect on cells

indel mutation

insertion or deletion of nucleotides

• effect on protein depends on size

• different forms or “alleles” of a gene have slightly different DNA sequences, these differences may or may not affect the protein that is made

• effect depends ion how many nucleotides are added or deleted

• non-multiples of three throw off the reading frame and cause a “frameshift”, which almost always leads to a premature STOP codon

• insertions'/deletions of 3,6,9,etc. may or may not have an effect, depending on what is gained or lost, and where

What if the effect of mutations in DNA locations other than protein-coding?

• have a less predictable effect

• mutations in a promoter could affect how much transcription occurs- or have no effect

• mutations in an intron could affect whether splicing works properly-or have no effect

what are the feature of a membrane system?

• allows fro compartmentalization of processes

• creates a “gatekeeper” to seperate internal and external environments

  • differences across membrane

• allows for establishment of concentration gradients which can be used to store and harness energy

• serves as a communication center to receive and send signals

what are membranes necessary for?

• cellular structure and function

• eukaryotic cels are surrounded by a plasma membrane and contain membrane-bound organelles

• membrane consist of lipids, proteins, and carbohydrates

fatty acid

long hydrocarbon “tail” with carboxyl group at one end, a “building block” of fats

steroid

includes 4 fused hydrocarbons rings

amphipathic

a molecules that is both hydrophilic and hydrophobic

what are phospholipids made up of?

• phospholipids are amphipathic with a hydrophilic head and a hydrophobic tail

• made up of: polar or charged group of some kind + phosphate + glycerol + 2 fatty acids

unsaturated

one or more double bonds in a tail

• because it’s not the max number of H atoms

• causes a kink in the tail → not as close together → doesn’t form as many VDW interactions

• kinks = more flexible membrane

saturated

all single bonds in tail

what forms membranes

• bilayers of phospholipids

• phospholipids are amphipathic, leading to the bilayer structure of a cell (and organelle) membranes

• the hydrophilic heads will interact with water and face the watery environment outside the cell and inside the cells

• the hydrophobic tails will not interact with water and will associate with each otehr inside the bilayer

phospholipid bilayer

forms membrane structure; hydrophobic core

cholesterol

a steroid that afects membrane fluidity

intergral membrane proteins

enter or completely span membrane

transmembrane proteins

type of integral protein that completely crosses the membrane

peripheral membrane proteins

associate with one side of membrane

glycolipids

lipid with covalent addition of carbohydrates

• functions include cell recognition and cell adhesion

  • happens on the outside

  • good for our immune system

how can membranes be different?

• different cell types have different phospholipid compositions and protein components

  • gives membranes different properties

• different organelles can have different components

• even the two leaflets of the bilayer itself can differ from one another in exact lipid components

glycoprotein

protein with covalent addition of carbohydrates

leaflets

one of the two layers in a bilayer

fluid

can bend and stretch without breaking

• membranes are fluid

what movements are favorable for phospholipids?

• lateral diffusion

• rotation

• flexing of tails

what movements are unfavorable for phospholipids?

flip from one leaflet to the other

how does fatty acid saturation influence membrane fluidity?

• saturated: less fluid

  • less moveable bc of how tight and amount of VDW interactions

• unsaturated: more fluid

  • double bonds causes kinks → tails cannot pack closely together, few VDM

where is cholesterol in the cell?

cholesterol, like phospholipids, is amphipathic and this proerty will orient the molecule within the membrane

how does cholesterol effect membrane fluidity?

cholesterol hydroxyl interacts with the polar head of a phospholipid; cholesterol’s rings and tail interact with the fatty acid tails. Thus, cholesterol holds adjacent phospholipids together and typically makes membranes less fluid

what substences cross the membrane, and how do they do so?

• lipid bilayer has limited permeability

• small hydrophobic molecules readily diffuse across the lipid protion of the membrane while others require membrane proteins for transport

how do memebranes help set up and maintain gradients?

limited permeability and the function of transports creates gradients

gradients

different concentrations on either side of the membrane

• gradients store potential energy

diffusion

net movement of particles from an area of high concentration to low concebtration

passive

does not require the input of external energy

• high → low

simple diffusion

very small, hydrophobic molecules readily diffuse across the lipid portion of the membrane down their concentration gradients

• no assistance needed

facilitated diffusion

molecules moving across the membrane via protein transporters or channels

how does water cross the membrane?

most of the water that enters cells does so by facilitated diffusion through proteins called aquaporins

osmosis

the diffusion of water across a semi-permeable membrane (like a cell membrane)

• water moving down it concentration gradient

  • free water concentration

• water moves towards regions of low free water concentration

• wtaer moves towards regions of high solute concentration

aquaporin

a channel protein for water

concentration

amount of solute in volume of solvent

amount of solute (moles)/ volume of solvent (L)

hypotonic solution

relatively lower concentration of solute

hypertonic solution

relatively higher concentration of solute

isotonic

if solute concentration are the same

osmolarity

a measure of the concentration of all solutes in a solution

• influences the direction water will move during osmosis

how do ions cross the membrane?

• ions cannot pass through the lipid portion of the membrane freely/readily

• ions must pass through membrane proteins to enter to enter or exit cells

  • must do facilitated diffusion