CMB405 Exam 2

density gradient centrifugation

density gradient centrifugation

method Messelsohn & Stahl used to figure out how DNA replicates

replication origin (ori)

specific site where replication begins

replication fork

junctions where 2 strands of DNA separate to allow replication of each strand

helicase

unwinds DNA, breaking H-bonds (DNA replication)

single stranded binding proteins

keep DNA unwound (single stranded)

gyrase

• in prokaryotes, moves ahead of replication fork;

• relieve torsional strain and help DNA unwind (similar to topoisomerase II in eukaryotes)

DNA polymerase

• enzyme that assists DNA replication • catalyze the polymerization of dNTPs alongside a ssDNA strand

DNA polymerase requirements

1. needs a free 3' OH to add new dNTP onto (synthesizes new strand from 5' → 3')

2. requires a template

3. synthesizes in antiparallel fashion

primer

oligonucleotide (short sequence) of DNA or RNA that "primes" replication by polymerase

leading strand

continuously synthesized; moves toward replication fork

lagging strand

discontinuous synthesis; has to wait for replication fork to open more; made in fragments

primase

adds RNA primer to lagging strand (does not require 3' end or primer to start)

DNA pol. III

uses RNA primer to start synthesizing new DNA strands by adding nucleotides

DNA pol. I

removes the RNA primer and replaces it with DNA

ligase

connects fragments of DNA in the lagging strand

beta clamp

identifies where pol. III will bind next (at end of RNA primer) on lagging strand

beta clamp loader

holds beta clamp and loads it onto the next RNA primer

polymerase chain reaction (PCR)

1. denaturing

2. annealing

3. extension

denaturing

• use heat to break apart H-bonds (dsDNA → ssDNA) • ~95℃

annealing

• primers provide 3' OH group for polymerase • ~50-60℃

extension

• DNA polymerase binds to primer and synthesizes more DNA (PCR)

• 72℃

taq polymerase

DNA synthesis enzyme that can withstand the high temperatures of PCR

telomerase

a reverse transcriptase that synthesizes telomeric repeat sequences at the ends of chromosomes from its own RNA template (extends telomeres)

structure of RNA

• has a 2' OH group • uracil (no thymine) • usually single-stranded • unstable → easily degraded • directional (5' → 3') • synthesized antiparallel to DNA

TATA box

a promoter DNA sequence crucial for transcription initiation

sigma factors

in prokaryotes, binds to RNA polymerase and directs it to the promoter region; important for initiating transcription

+1 start site

where the first nucleotide of the RNA strand is made

transcriptional bubble

formed where RNA polymerase interacts with DNA and pulls apart the two strands

bacterial transcription

• one type of RNA polymerase • promoter structure (-35, -10) • multiple sigma factors

eukaryotic transcription

• multiple types of RNA polymerases • TATA box slightly further upstream

TBP

TATA binding protein (one of the transcription factors)

TFIIB

marks +1 start site

TFIIA

• initiates the pulling apart of DNA (winds DNA around polymerase)

• helps bend/melt DNA (transcription)

TFIIH

helicase; ahead of polymerase; unwinds DNA (transcription)

carboxy terminal domain

• protein tail of RNA polymerase • can be phosphorylated (help recruit other enzymes) • allows factors involved in mRNA processing to "ride along" with RNA polymerase ↳ as RNA is being made, 5' cap, splicing, and poly A tail also ocurring

terminator sequence

• sequence of bases at the end of a gene that signals the RNA polymerase to stop transcribing • in DNA • inverted repeats that get incorporated into RNA

transcription termination

induced when the hairpin structure in RNA forms, causing RNA polymerase to release the RNA transcript and fall off DNA

mRNA processing

1. 5' cap

2. poly A tail

3. splicing

5' cap

• derivative of guanosine • 5' to 5' linkage • protect 5' end from degradation (nucleases can't degrade) • shuttle mRNA out of nucleus • aids in translation

poly A tail

• string of adenines added onto the 3' end of the RNA • protects 3' end from exonucleases • transport out of nucleus • aids in translation • longer tail = longer half-life (confers stability)

poly A tail polymerase

adds a chain of adenines to the RNA

splicing

• pre-mRNA is converted to mRNA by removing intron sequences • introns are removed and exons are brought together

spliceosome

controls splicing

U1

spliceosome that recognizes 5' splice site

U2

spliceosome that recognizes branch point

U4, U5, U6

spliceosomes that interact with all 3 splicing consensus sequences

splice junction sequences

one at 5' end of RNA, one at 3' end, and branch point somewhere in the middle; conserved sequences

group II introns

• intricate structure • can splice themselves out; do not need spliceosome

alternative splicing

splicing of introns in a pre-mRNA that can occur in multiple ways, leading to different mRNAs that code for different proteins

translation

• RNA → protein • occurs in the cytoplasm (outside of the nucleus) in eukaryotes • in prokaryotes, can occur as transcription is happening

peptide bond

links amino acids together (know different between these and phosphodiester bonds)

N terminus

the end of a polypeptide or protein that has a free amino group

C terminus

the end of a polypeptide or protein that has a free carboxyl group

levels of protein structure

primary, secondary, tertiary, quaternary

AUG

start codon

untranslated region

UTR (5' and 3')

properties of the genetic code

universal, non-overlapping, contains punctuation, degenerate (redundant)

synonymous (silent) mutation

does not change the amino acid sequence

nonsynonymous (missense) mutation

causes an amino acid substitution

nonsense mutation

creates a premature stop codon

frameshift mutation

alters the normal reading frame of the mRNA; insertion or deletion of nucleotides

tRNA

• act as adaptors to bring amino acids to their codon • anticodons form complementary base pairs with mRNA codons (antiparallel to mRNA)

wobble position

• base pairing is more flexible and can sometimes tolerate mistakes • depends on codon/anticodon and tRNA

large ribosomal subunit

50S

small ribosomal subunit

30S

aminoacyl-tRNA synthetase

attach amino acids to specific tRNAs

aminoacyl site

where the charged tRNA is recruited

peptidyl site

site of peptide linkage

exit site

site where tRNAs exit the ribosome

shine dalgarno sequence

• translation consensus sequence found in prokaryotes • uses rRNA to find consensus sequence on mRNA

initiation factor (IF 1-3)

bind to the small subunit and help ribosome bind to mRNA

elongation factor (EF)

one of multiple proteins that facilitates the lengthening of a polypeptide during protein synthesis

EF-Tu

elongation factor that recruits the next charged amino tRNA into the A site

EF-G

helps translocate the ribosome down 3 nucleotides

ribosome inhibitors

neomycin/gentamycin (aminoglycoside); tetracycline; chloramphenicol

mechanisms of ribosome inhibitors

• prevent ribosome assembly • block A site of the ribosome; prevent new tRNA from reading codon • block peptidyl transfer from P to A tRNA (block peptide formation)

eukaryotic protein synthesis inhibitors

puromycin and ricin

housekeeping genes

constitutive genes that are required for the maintenance of basic cellular function

structural genes

code for proteins used in metabolism, biosynthesis, structural role

regulatory genes

products of these genes interact with other sequences and affect transcription/translation

regulatory elements

DNA sequences that are not transcribed but affect expression of other sequences

operon

group of genes controlled by a single promoter

operator

DNA element that binds repressor

repressor

binds to operator and prevents RNA polymerase from transcribing genes

lac Z

codes for β-galactosidase; breaks lactose into galactose and glucose

lac Y

codes for permease

permease

allows lactose to enter the cell

catabolite activator protein (CAP)

• protein regulated by cAMP • recruits RNA polymerase to promoter (lac operon)

• aka cyclic AMP receptor protein

inducer

binds to and deactivates repressor

zinc finger

DNA binding protein motif that contains zinc ion

helix-loop-helix

DNA binding protein motif that has 2 α-helices separated by a loop

leucine zipper

contains regularly spaced leucine residues (don't need to know spacing, but it's every 7 amino acids)

DNA binding protein motifs

• helix-turn-helix • zinc fingers • leucine zippers

transcription factor functional domains

• DNA-binding domain • activation domain • repression domain

enhancer

DNA sequence bound by activator protein (eukaryotic regulatory element)

silencer

DNA sequence bound by repressor protein (eukaryotic regulatory element)

kozac sequence

eukaryotic consensus sequence surrounding the start codon

release factor proteins

bind to the stop codon and release the ribosome from mRNA, terminating translation