BIO130

A detailed set of cards prompting BIO130 students to recall information for the midterm exam

Genome

entirety of an organism’s genetic info (all the DNA in a cell)

sequencing a genome

determining the order of all the bases in the DNA

human genome has ~__ base pairs per genome

3 billion

in a standard cell, you have _ genomes

2 (one from each parent)

genome size is or isn’t always correlated with complexity?

isn’t

_% of your genome is repetitive DNA

50

less than _% of your genome encodes protein

1

nonrepetitive DNA (~50%)

includes sequences that tell the cell how much RNA to make and what to transcribe

nonrepetitive DNA includes __ and __

introns (transcribed then spliced out) and exons (transcribed and translated)

mobile genetic elements

sequences that are sometimes spliced out of the DNA, sometimes copied, sometimes pasted back in

LINEs

Long Interspersed Nuclear Elements

A type of mobile genetic element

>= 500 base pairs

SINEs

Short Interspersed Nuclear Elements

noncoding

Type of mobile genetic element

< 500 base pairs

retrotransposons

transcribed (made into RNA)

DNA-only transposons

not transcribed into RNA

Genome occupies a large portion of the cell volume

false

DNA packaging in prokaryotes

• have no nucleus

• but DNA is in compact structure called the nucleoid

Chromosome Painting Hybridization

a technique that uses many fluorescent probes, one for each chromosome, so you can visualize which are present in a sample. (FISH with a lot of probes)

Fluorescence in Situ Hybridization (FISH)

A diagnostic technique for testing the presence of a particular sequence

• probe DNA binds to the sequence you want to detect (bc it’s antiparallel complementary)

• use fluorscent dye that attaches to the probe

• separate sample DNA strands so that they’re available for the probes to attach

• once it cools back down look at the sample. If fluorescence, then probe sequence had been able to bond to the target sequence confirming the target’s presence in the sample.

__ pairs of chromosomes in humans

23

chromatin

single, long, linear DNA molecule and associated proteins

• “beads on a string”

chromatid vs chromosome

30 nm fiber

name of the structure where chromatin fiber is packed into nucleosomes

histones

• small proteins rich in lysine and guanine

• have positive charges that neutralize DNA’s negative charge

• is an octamer core with a pair of each 4 core histone proteins (H2A, H2B, H3, H4)

• there’s also H1, the linker histone

What does H1, the linker histone do?

clips the DNA onto the histone octamer

nucleosome core particle vs nucleosome

nucleosome core particle = core histone + DNA around it

nucleosome = nucleosome core particle + H1 + linker DNA

sequence-specific clamp proteins and cohesins

involved in forming chromatin loops

condensins

as cells enter mitosis, condensins replace most cohesins to form double loops of chromatin to generate compact chromosome

chromatin remodeling complexes/histone modifying enzymes

proteins that can make changes in chromatin structure and alter access to DNA for replication or transcription

heterochromatin vs euchromatin

h: highly condensed → gene expression suppresses

• meiotic/mitotic chromosomes

• centromeres and telomeres

• time spent highly condensed varies (constitutive vs facultative (more temporary))

e: relatively non-condensed → genes tend to be expressed

• degree of condensation varies

• level of activity varies (quiescent vs active)

  • active = doing something w the DNA

DNA synthesis is always __conservative

semi

DNA polymerase

catalyzes DNA synthesis (adding on base pairs to template strand)

3 rules for DNA synthesis:

1. DNA is antiparallel and complimentary

2. new DNA is synthesized from 5’ - 3’

3. the template is read 3’ - 5’

origin of replication

starting point for bidirectional growth

• easy to open, A-T rich (only 2 H bonds)

• recognized by initiator proteins that bind to the DNA

Bacteria have 1, Eukaryotes have multiple

prokaryote vs eukaryotic cell

P:

• no nuclei

• single-celled

• no membrane-bound organelles

• smaller

• less DNA

• (bacteria and archaea)

E:

• nuclei

• single OR multi-celled

• membrane-bound organelles

• larger

• more complex

• (plants, fungi, animals)

mitochondria

uses oxygen to generate ATP through aerobic respiration

endosymbiont, started as ectosymbiont

3 lines of evidence that supports endosymbiont hypothesis for origin of mitochondria and chloroplasts

1. they have their own cell walls that resemble that of prokaryotes

2. they have remnants their own genomes and genetic systems that resemble that of prokaryotes

3. they have their own protein and DNA synthesis components

general attributes of a model organism

1. readily available

2. tractability - ease of maniipulation/modification

3. we understand its genetics

4. rapid development with short life cycles

5. small adult (reproductive) size

(like a rat!)

3 types of RNA that eukaryotes and prokaryotes have in common

Messenger RNA (mRNA) - blueprint

Transfer RNA (tRNA) - brings amino acids

Ribosomal RNA (rRNA) - structural for ribosome, break + form proteins

nucleic acids

genetic material in a cell

DNA = deoxyribonucleic acid

RNA = ribonucleic acid

made up of nucleotides

nucleotide structure

phosphate group (backbone) - attached to 5’ C of sugar

nitrogenous base - attached to 3’ C of sugar

pentose sugar (scaffold for base)

two types of bases

pyrimidines - “U C The pyramids” → Uracil, Cystine, Thymine

purines - adenine, guanine

(side chains make bases different from each other)

H-bond together when base-pairing

nucleoside vs nucleotide

nucleotide if has at least one P, nucleoside if not (just base + sugar)

3 forces that keep DNA strands together

1. Hydrogen bonding between bases

2. hydrophobic interactions (bases want to get away from water, press in closer)

3. can der Waals attractions (molecules closely packed)

structure of amino acid

carboxyl group, R-group, amine group, hydrogen → connected to central C

initiator proteins

binds to replication origin to help DNA helicase bind (it destabilizes the AT-rich sequence)

helicase-loading protein

helps helicase load onto the DNA then leaves

helicase

unwinds and separates strands

• predominant helicase moves 5’-3’ along the lagging strand template

single-strand binding proteins

keep DNA strands separated after they are separated by helicase

RNA Primers and DNA Primase

Primase makes primers, which is a short sequence of nucleotides with a free 3’OH. DNA polymerase requires a “bound primer” to begin adding nucleotides to the template strand.

Primase proceeds in the 3’→5’ direction

Primosome

DNA Polymerase + DNA Primase

Sliding clamp

holds polymerase onto the DNA so it doesn’t float away

DNA ligase

removes RNA primer and replaces it with a DNA sequence

• is a “DNA repair system”

This is important for linking the Okazaki fragments together

Replisome

A type of “molecular machine” - all the proteins that work together to replicate DNA

okay this is a hard one…recount the process of DNA replication

initiator protein → helicase-loading protein → helicase unwinds strands (single-strand binding proteins needed to keep them separated) → DNA polymerase + DNA primase = primosome (sliding clamp needed to keep polymerase from floating away) → leading strand and lagging strand synthesis are simultaneous (2 replication forks moving in opposite directions)

topoisomerase

solves the problem of supercoils that form when helicase unwinds DNA. It makes a cut to release tensions then seals the cut.

What is the issue in DNA replication concerning the ends of linear chromosomes?

The lagging strand would miss information (if not for the solution that exists) because primase isn’t good at putting a primer at the very end, and once the 3’-most primer gets removed there’s no 5’ end to add onto—missing DNA!

Telomerase

A solution for the lagging strand’s problem with missing DNA at its ends.

• Telomerase uses an RNA template to make a DNA complementary copy

• it adds a repetitive sequence to the 3’ end of the parental strand (lagging strand template) → it’s so long now that primase CAN be placed and polymerase can come in and fill the gap we had earlier

Telomerase is not abundant in __ cells

somatic (connects to aging!)

connect telomerase to aging/cancer

Telomerase is what adds telomeres to the ends of linear chromosomes. Telomerase is abundant in stem and germ-line cells, but NOT in somatic cells. So, loss of telomeres occurs normally during DNA replication, and limits the number of round of cell division.

on the other hand, cancer tells have high levels of telomerase and so replicate abundantly.

Fill in the blanks with numbers:

RNA polymerases typically have an error rate of about 1 in __. DNA polymerases are only about 1 in __. The human genome (3×10^9) is only changed by about _ nucleotides every time a cell divides!

10^4, 10^9, 3

What are the two mechanisms in place that ensure accurate DNA synthesis?

1. 3’ to 5’ exonuclease

2. strand-directed mismatch repair

3’-5’ exonuclease

is on the DNA polymerase’s exonuclease (“editing”) site. Removes incorrectly placed nucleotides. “Proofreads” DNA.

strand-directed mismatch repair (in eukaryotes)

A DNA replication error repair process (for if proofreading fails). It is initiated by the detection of the distortion in geometry of the double helix generated by mismatched base pairs

(for prokaryotes: detect unmethylated adenines, because parental strand have them but not new strands)

DNA can be damaged by:

• oxidation

• radiation

• heat

• chemicals

• and more

Pyrimidine Dimers

when UV radiation causes covalent bonds when they’re not supposed to between adjacent bases

Depurination

a base is lost spontaneously because a water molecule hit the DNA strand at the wrong time/energy/space/spot

Deamination

An amine is spontaneously bumped (by a water molecule) from the cytosine and it becomes a uracil instead

The two general mechanisms of DNA repair of incorrect DNA:

1. Base Excision Repair (BER)

2. Nucleotide Excision Repair (NER)

Base excision repair (BER)

Fixes one nucleotide at a time. Following the example of the image:

• Uracil DNA glycosylase comes in a removes the incorrectly placed uracil

• two enzymes (AP endonuclease and phospodiesterase) come in and remove the sugar phosphate backbone

• so then polymerase can come in and repair

Nucleotide Excision Repair

Fixes a couple incorrectly placed nucleotides at a time.

• excision nuclease comes in, makes two cuts

• a helicase removes a strand

• polymerase comes in and fixes the strand

The two methods of DNA repair of double-stranded breaks:

1. Nonhomologous end joining (NHJ)

2. Homologous recombination (HR)

Nonhomologous end joining (NHJ)

A method of repair for double stranded breaks in DNA. Nuclease trims the strand then joins it together with a special DNA ligase. “quick and dirty”, used in emergency in cell division to make sure daughter cells have the right number of chromosomes.

Homologous recombination (HR)

The more accurate method (but takes longer) of DNA repair of double stranded breaks. Uses info from the homologous chromosome.

Gene

The entire nucleic acid sequence necessary for the synthesis of a protieins or RNA. “segments of DNA that are transcribed into RNA”

ssDNA

single stranded DNA (the template for RNA)

phosphodiester bonds

bonds between nucleotides (in DNA and RNA)

sigma factor

A protein required in transcription for bacteria. It binds to RNA Polymerase core enzyme, searches for the promoter sequence on the DNA and binds to it. Once transcription begins, it’s released.

RNAP Holoenzyme

RNAP core enzyme + sigma factor

Promoter

The sequence of DNA where transcription starts for bacteria

• where sigma factor binds

• commonly at ~ -10 and ~ -35

Describe how terminator sequences work in bacteria

the end of a gene contains a lot of Gs and Cs, followed by As and Ts. The strong base pairing of the Gs and Cs gets in the way of the RNA Polymerase (“hairpin” structure).

primary or pre- mRNA transcript

the full mRNA transcript, including introns. but doesn’t have a 5’ cap or 3’ poly-A tail yet.

UTR

Untranslated regions (at the end of mRNA). They do not code for proteins.

translation and transcription are coupled in bacteria or eukaryotes?

bacteria

What is the start codon in mRNA for eukaryotic translation?

AUG

name what the three different RNA polymerases are mainly used for transcribing

RNAP I: most rRNA genes

RNAP II: all protein-coding genes

RNAP III: tRNA genes

carboxyl terminal domain tail (CDT)

special tail of the largest subunit of RNAP II. Tandem repeats of 7 amino acids. Allows transcription and RNA processing to be coupled by undergoing dynamic phosphorylation and recruiting proteins involved with capping, polyadenylation, and splicing.

transcription factors

Proteins required by RNAP to help position them at the promoter (similar to the sigma factor in bacteria)

TATA box

A common element (promoter sequence), bound ~ 30 bps upstream from the start site for transcription. It helps position RNAP II and general transcription factors.

TFIIH

A transcription factor that helps separate DNA strands and phosphorylates CTD

(can remember that “H” is for “Helicase”)

The 4 steps in eukaryotic transcription initiation (this is pretty detailed, but something we have to know)

1. TBP, a subunit of TFIID, binds to the TATA box promoter in the minor groove, bending and distorting the DNA

2. this attracts other transcription factors, which help orient and bind RNAP II to the DNA

3. the helicase activity of TFIIH uses ATP to pry apart DNA strands of transcription start site

4. TFIIH also phosphorylates the CTD of RNAP II, activating it so that transcription can begin

exonucleases

proteins that chop off nucleotides one at a time from nucleic acids

• protected against by poly-A tail and 5’ capping

describe how a intron is removed

1. Branch point A attacks the 5’ splice site

2. 3’ end of one exon connects with 5’ end of the other

3. exon junction complex added (like glue)

spliceosome

enzyme that catalyzes splicing

How it works:

• takes the 2’ oxygen off the ribose and forms a phosphodiester bond with the 5’ end of the intron

• that frees up a 3’ OH at the end of exon 1, and so a bond is formed with the 5’ end of exon 2

poly-A binding protein

Proteins added to the poly-A tail that make it hard for 3’ exonucleases to chop of the Adenines.

poly-A polymerase

a protein involved in RNA processing that adds an poly-A tail to mRNA

• does NOT bind to CTD

• does NOT require a template

snRNPs

proteins that make up the spliceosome. Recognizes splice-sites of mRNA, helps create the lariat. Made of snRNAs bound to a protein.

alternative splicing

pre-mRNAs can be spliced in multiple ways to create different versions of a protein from one gene (eukaryotes)

redundancy

most amino acids are encoded by multiple codons

3 types of substitution mutations:

silent: codon still refers to same amino acid

missense: codon refers to different amino acid

nonsense: codon changed to a premature STOP codon

other than substitution, what are the other two types of mutations?

1 nucleotide-pair insertion or deletion

• changes reading frame (really bad!)

3 nucleotide-pair deletion

• causes a missing amino acid