BIS2A SS1 Week 5

cancer cells and telomerase

cancer cells have really high telomerase activity, so they never senesce/die and lose “important” DNA

plasmid vs chromosome

what are some examples of essential genes

DNA replication/repair, membrane creation, transcription, translation, cell cycle regulating genes

3 ways that a cell can repair a mistake

1. repair the mistake by proofreading by pol III

2. DNA pol can leave the mistake behind and other enzymes can repair it

3. SOS polymerase in bacteria (last resort)

proofreading

when pol III detects a mistake, it can go back, cleave the bond it made, and replace the nucleotide with the correct nucleotide (exonuclease activity)

occurs in certain DNA polymerases and NO RNA polymerases

why doesn’t RNA polymerase proofread?

RNA is made and degraded, so mistakes only result in a few bad proteins

DNA mutations are permanent and can be passed down (need proofreading)

why is flu/COVID mutagenic?

they are RNA viruses, so the RNA polymerases are highly inaccurate

results in mutations in antigens to avoid immune system detections

mismatch repair (single strand repair)

replacing one wrongly inserted nucleotide with the correct one

done by DNA pol I; more local repair

excision repair (single strand repair)

removes a whole region of DNA containing mistakes and codes it again

common with frameshift mutations

done by DNA pol I

DNA methylation

used to recognize which strand is the daughter strand and which is the parent strand

can recognize which strand contains the mistake and repair it based on the methylation state

only works on repair enzymes before methylation enzyme

is the parent or daughter strand unmethylated before methylation enzyme?

daughter stand is unmethylated BEFORE the methylation enzyme comes

therefore, the mistake is in the unmethylated strand

what two processes occur if both strands are methylated and there is damage?

photoreactivation or recombination repair

photoreactivation

photoreactivation genes use the energy of the light and can leave damage cause by UV rays by cleaving C-C or T-T dimers that arise

can also repair nicks or breaks in DNA

recombination repair

using the homolog chromosome as a template for repair

eukaryotes have the advantage here as we are diploid; prokaryotes can only do this during replication

SOS polymerase

the last line of defense for bacterial cells for death, equivalent to a cell cycle stop in eukaryotes (ex. blocked replication fork)

stuffs a bunch of random nucleotides (results in a lot of mutations bc no proofreadings)

double strand breaks

when chromosomes break, need to repair the open ends

non-homologous end joining (NHEJ)

putting together breaks regardless of the chromosome (joining together the wrong chromosomes)

microhomology-mediated end joining

more homology than NHEJ but still on the wrong chromosome

homologous recombination (HR)

ends of DNA are attacked by a single strand; using homolog chromosome to repair double strand breaks

process of HR with linear into linear DNA

DNA with free ends invade (cell doesn’t like), 3’ end of that DNA is chewed up by recombination enzymes, DNA is replaced (reciprocal recombination)

2 recombination events

RecA

binds all over the DNA with free ends to align the DNA for the enzyme to complete replacement

recombination frequency

the closer genes are linked, the lower their recombination frequency is (more likely to be inherited together)

recombination is random at 50%

HR of linear into circular DNA

2 recombination events

HR of circular into circular DNA

1 recombination event

why is recombination important?

gene mapping, eukaryotic meiosis

what are the 3 ways that a bacteria can move around/swap DNA?

transduction, transformation, conjugation

transduction

phages can move DNA from one bacterial cell to another

transformation

cell can take up DNA from the outside environment (like plasmids)

conjugation

F+ (sex pilus) can replicate and transfer a plasmid to an F- bacteria (makes it F+)

central dogma

DNA —> RNA (transcription) — nucleus

RNA —> protein (translation) — cytoplasm

gene

information containing DNA that can code for proteins

transcription

DNA (gene) —> mRNA

only use a small segment of one strand of DNA (template strand)

template strand

the strand the RNA polymerase reads to make mRNA

coding/nontemplate strand

the strand complementary to the template strand

replacing the T’s with U’s gives you the mRNA

RNA polymerase

the only polymerase involved in transcription (doesn’t require primers, helicases, etc.)

in bacteria, interacts with sigma and backbone

in euks interact with transcription factors

in which way is the template strand read? the mRNA transcribed?

template strand read from 3 to 5

mRNA transcribed 5 to 3

promoter sequence

the site where the RNA polymerase binds

determines which strand of DNA is read

promoter consensus sequence

a sequence that is common throughout all bacteria, where sigma binds

sigma (bacterial intiation)

transcription factor that helps RNA polymerase bind to the promoter consensus sequence (-10, -35 promoter)

TATA box (eukaryotic/archaea promotor)

TATA binding protein binds to the TATA box, recruits enzymes to help RNA pol bind

what bases are the promoters rich in?

A and T

transcription start/initiation site

where transcription starts

overlapping/adjacent to the promoter

transcription stop/termination site

where transcription ends

what are the two types of transcription termination in bacteria?

factor independent and factor dependent termination

factor independent termination

hairpin and U sequence cause the RNA polymerase to hop off

size of hairpin + length of U sequence determine terminator effectiveness

factor dependent termination

rho protein binds to mRNA and knocks the RNA polymerase off

operons (bacteria)

polycistronic stretches of DNA, controlled by one promoter and transcription stop site

how to regulate expression of genes

adding weak terminators (hairpins) between genes allows downstream genes to be expressed less

all types of passive control of bacterial genes

promoter strength/location, termination strength/location, sigma factor

immature RNA

in euks — mRNA that is in nucleus and undergoes post-transcriptional changes to be ready for translation

what are the 3 post-transcription modification

add 5’ G cap and 3’ poly-A tail to stabilize RNA

splicing — remove introns and join exons (can make multiple proteins from same stretch of DNA)

translation

mRNA —> protein

initiation

finding the start codon after recognizing the G cap

translocation

ribosome translocates to next codon; costs 2 GTP

elongation

creating the amino acid sequence in the APE sites

termination

reading the stop codon

brings in the release factor in A site to release polypeptide chain

start codon

AUG; codes for methionine

when looking at a sequence of bases, always look for the start codon

stop/nonsense codons

stop codons don’t code for amino acids

what is needed for translation?

mRNA, amino acids, ribosome, tRNA

tRNA structure

one end has anticodons that are complementary to codons (determines if correct AA being added)

other end carries the AA

aminoacyl-tRNA synthetase

enzyme that consumes ATP to attach the amino acid to the tRNA to make it into charged tRNA

ribosome

makes proteins; composed of a small and large subunit that sandwich mRNA

made of rRNA and proteins

A site (aminoacyl site)

entry site for the charged tRNA

peptidyl site (P site)

forms the peptide bond between the amino acids

exit site (E site)

where the uncharged tRNA exits once the peptide bond is formed

ribosome binding site

the site overlapping/next to translation start on the protein-coding sequence where the ribosome binds

how much does it cost to make one peptide bond

costs 1 ATP (aminoacyl-tRNA synthetase) and 2 GTP

gene expression

the expression of genes can be regulated by a variety of factors

regulatory region

a site before the promoter that can also affect gene expression

alleles

different variants of the same gene

what prevents RNA polymerase from binding to the promoter?

1) promoter sequence

2) prevent binding with other factors

strong promoter

a promoter where RNA polymerase can bind without the help of transcription factors

weak promoter

a promoter where RNA polymerase needs help of TFs to bind

sigma protein transcription control (bacteria)

there are multiple sigma factors that control specific genes and bind RNA pol at different strengths

what promoter RNA pol binds to depends on the sigma factor and its strength

transcription factors

proteins that make the promoter site available to RNA polymerase

activators

turns on gene expression for weak promoters (positive gene regulations)

repressors

turns off gene expression for strong promoters (negative gene expression)

what are the layers of gene regulation

1) promoter strength (weak or strong)

2) transcription factors (activator or repressor)

3) regulating transcription factors

regulation of transcription factors

allosterically regulated by effector molecules

inhibitor (effector)

binds to activator to deactivate it (decreases transcription)

co-repressor (effector)

binds to repressor to activate it (decreases transcription)

inducers (effector)

binds to activator to activate it OR binds to repressor to deactivate it

increases transcription

major difference between prokaryotic and eukaryotic genomes

eukaryotic organisms often have a lot more protein targeting/secretion and cell structure genes (organelles/endomembrane system)

in humans, which types of cells do we need to be careful in replicating?

germ line cells, as they are passed down to offspring

sanger sequencing

enzymatic sequencing that uses dideoxynucleoside triphosphates (ddNTP) and relies on RNA polymerase’s inaccuracy

when ddNTP is incorporated, replication stops (doesn’t have a 3’OH)

how is the DNA sequence from sanger sequencing read?

using gel electrophoresis, read off products of increasing length by nucleotide to read the sequence of the longest strand of DNA

fluorescently labeled nucleotides

the more common technique, where readings of florescent nucleotides can map out the sequence

major problem with genome sequencing

works well in bacteria with only one allele

problem is repeats in sequences makes it hard to order genes and completely assemble them

BLAST

has a +1, +2, +3 reading frame, which allows you to map out the codons in DNA from 5’ to 3’

once DNA is mapped out, can compare DNA to see which protein/enzyme it is

can also do the complement strand (-1, -2, -3) because can’t determine template or coding strand

cDNA

using all message and reverse transcriptase to get all coding regions of DNA

what is the difference between a mutation in a haploid organism and a polyploid organism?

mutations in haploid organisms are much more visible in comparison to polyploid organisms

do not have chromosomes to make up for any lack of critical gene expression