Unit 6: Gene Expression & Regulation Flashcards
Frederick Griffith (1928) Finding
living R bacteria transformed into
deadly S bacteria by unknown, heritable substance
Avery, McCarty, MacLeod (1944)
Tested DNA, RNA, & proteins in heat-killed pathogenic bacteria
Discovered that the transforming agent was DNA
Bacteriophages
virus that infects bacteria; composed of DNA & protein
Hershey and Chase (1952)
DNA entered infected bacteria -> DNA must be the genetic material!
Chargaff’s Rules:
DNA composition varies between species
Ratios: %A = %T and %G = %C
C & T Pyrimidine , A & G = Purines
Rosalind Franklin (1950’s)
X-ray crystallography = images of DNA
Provided measurements on chemistry of DNA
James Watson & Francis Crick (1953)
Discovered the double helix
DNA = Double Helix: Backbone & Rungs
“Backbone” = sugar + phosphate
“Rungs” = nitrogenous bases
Nitrogenous Bases
Adenine (A), Guanine (G)
Thymine (T), Cytosine (C)
A-T, C-G pure as gold,
What bonds are between base pairs of the 2 strands holding together molecule like zipper?
HYDROGEN BONDS
DNA strands = Antiparallel
Antiparallel, One strand (5’ -> 3’), other strand runs in opposite,
upside-down direction (3’ -> 5’)
How is DNA packaged? - Histones
the wrapping of DNA affects how genes are turned on or off ex: when chromosomes are tightly packed, it makes it more difficult for the transcription process to occur effectively. DNA is usually bound around the histones (supercoiled = does not express)
Prokaryotic DNA
Double-stranded
Circular
One chromosome
In cytoplasm
Supercoiled DNA
(nucleoid)
No histones
Eukaryotic DNA
Double-stranded
Linear
Usually 1+ chromosomes
In nucleus
Chromatin = DNA wrapped
around histones (proteins)
DNA Replication:
Making DNA from existing DNA
Meselson & Stahl (DNA Replication)
Replication = semiconservative & occurs 5’ -> 3’
DNA Replication = Semiconservative meaning
2 strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original strand and one new strand
Helicase
unwinds DNA at origins of replication & creates replication forks
Primase
Adds RNA primer to start replication
DNA polymerase III
adds complimentary nucleotide bases covalently to leading strand (new DNA is made 5’ → 3’)
Okazaki Fragments:
Short segments of DNA that grow 5’->3’ that are added onto the Lagging Strand
DNA pol I
replace RNA primer w/ DNA
DNA Ligase
joins 3’ end of DNA that replaces primer to rest of leading strand seals together fragments of lagging strand
Topoisomerase
relieves overwinding strain ahead of replication forks by breaking, swiveling, rejoining DNA strands
Lagging strand grows?
in 3’→5’ direction by the addition of
Okazaki fragments
Gene Expression
process by which DNA directs the synthesis of proteins (or RNAs)
one gene-> one RNA molecule (which can
be translated into a polypeptide)
Central Dogma
DNA -> RNA -> Proteins
TranSCRIPTion
DNA -> RNA
TranSLATion
RNA -> Proteins
Ribosome =
Site of translation
Difference between flow of genetic information between eukaryotes & prokaryotes
Prokaryotes = no nucleus so DNA can legit touch RNA & Ribosome
Eukaryotes = have nucleus containing RNA and DNA but ribosome = in cytoplasm
One gene = how many RNA molecules?
1
DNA
Nucleic acid composed of
nucleotides
Double stranded
Deoxygenated (Deoxyribose=sugar)
Thymine
Template for individual
RNA
Nucleic acid composed of
nucleotides
Single-stranded
Ribose=sugar
Uracil
Many different roles!
pre-mRNA
precursor to mRNA, newly transcribed and not edited
mRNA
edited version; carries the code from DNA that specifies amino acids
tRNA
carries a specific amino acid to ribosome based on its anticodon to mRNA codon
miRNA/siRNA
micro/small interfering RNA; binds to mRNA or
DNA to block it, regulate gene expression, or cut it up
mRNA (5’ → 3’)
complementary to
DNA template bc template (3’ -> 5’)
mRNA triplets (codons)
code for amino acids in
polypeptide chain
Redundancy Genetic Code
1+ codons
code for each of 20 AAs
Reading frame (genetic code)
groups of 3 must be read in correct groupings
Transcription unit
stretch of DNA that codes for a polypeptide or RNA (ex: tRNA, rRNA)
RNA polymerase
Separates DNA strands and transcribes mRNA
mRNA elongates in 5’ → 3’ direction
Uracil (U) replaces thymine (T) when pairing toadenine (A)
Attaches to promoter (start of gene) and stops at terminator (end of gene)
Initiation (Transcription) in Bacteria
RNA polymerase binds directly to promoter in DNA
Initiation (Transcription) in Eukaryotes
TATA Box
Promoter Region
Transcription Factors
TATA box + Promoter region (initiation, transcription) Euk
DNA sequence (TATAAAA) in promoter region upstream from
transcription start site
Upstream DNA
toward the 5’ end of the coding strand for the gene
Downstream DNA
toward the 3’ end
Coding Strand
determines the correct nucleotide sequence of mRNA
not in transcription
3’ -> 5’
complimentary nucleotide sequence
Template strand
base for mRNA transcription
antisense strand, non coding, take part in transcription & Help formation of mRNA.
5’ to 3’ no complimentary sequence
Antiparallel nature of DNA
2 strands, each with a backbone of alternating phosphate & sugar groups
Strands run in opposite directions (5’ -> 3’ & 3’ -> 5’)
Transcription factors (initiation, transcription) eukaryotes
must recognize TATA box before RNA polymerase can bind to DNA promoter
Transcription Initiation complex
Transcription Factors + RNA Polymerase =
Elongation (Transcription )
RNA polymerase adds RNA nucleotides to the 3’ end of the growing chain (A-U, G-C) As RNA polymerase moves, it untwists
DNA, then rewinds it after mRNA is made
Termination (Transcription) Prok
RNA polymerase transcribes a terminator sequence then mRNA and polymerase detach NOW mRNA ready to use
Termination (Transcription) Euk
polyadenylation signal sequence then mRNA and polymerase detach NOW called pre-mRNA
Eukaryotic cells modify RNA after transcription
5’ cap
3’ poly-A tail
5’ cap
(modified guanine)
3’ poly-A tail
(50-250 A’s) are added
Functions for 5’ cap & 3’ poly-A tail
- Export from nucleus
- Protect mRNA from enzyme degradation
- Attach mRNA to ribosomes in cytoplasm
RNA Splicing
Pre-mRNA has introns (noncoding sequences) &
exons (codes for amino acids)
Splicing = introns cut out, exons joined together
RNA splicing (cont.) snRNPs
snRNPs join with other
proteins to form a spliceosome
RNA Splicing (cont. Spliceosome)
catalyze (makes faster) the process of removing introns &
joining exons
Ribozyme = RNA acts as enzyme (catalytic role)
Why do we have introns?
Some regulate gene activity
Alternative RNA Splicing: produce different combinations of exons
One gene can make more than one polypeptide!
20,000 genes → 100,000 polypeptides
Components of Translation
mRNA = message
tRNA = interpreter
Ribosome = site of translation
tRNA
Transcribed in nucleus. Specific to each amino acid. Transfer AA to ribosomes
Anticodon
pairs w/ complementary mRNA codon
Wobble (translation)
Base-pairing rules between 3rd base of codon & anticodon are
not as strict.
Aminoacyl-tRNA-synthetase:
enzyme that binds tRNA to specific amino acid
Ribosomes
rRNA + proteins,made in nucleolus
2 subunits: Active sites: A site: holds AA to be added & P site: holds growing polypeptide chain
E site: exit site for tRNA
Ribosome active sites
A site: holds AA to be added
P site: holds growing polypeptidechain
E site: exit site for tRNA
Initiation (tranSLATion)
Small subunit binds to start codon (AUG) on mRNA
tRNA carrying Met attaches to P site
Large subunit attaches
Elongation (tranSLATion) Codon Recognition
tRNA anticodon matches codon in A site
Elongation (tranSLATion)Peptide bond formation:
AA in A site forms bond w/ peptide in P site
Elongation (tranSLATion): Translocation
tRNA in A site moves to P site; tRNA in P site moves to E site (then exits)
Termination (tranSLATion):
Stop codon reached & translation stops
Release factor binds to stop codon; polypeptide = released
Ribosomal subunits dissociate
Protein Folding
During synthesis, polypeptide chain coils & folds spontaneously
Chaperonin: protein that helps polypeptide fold correctly
Post-Translational Modifications
Attach sugars, lipids, phosphate groups, etc.
Remove amino acids from ends
Cut into several pieces
Subunits come together
Free ribosomes:
synthesize proteins that stay in cytosol & function there
Bound ribosomes (to ER):
make proteins of
endomembrane system (nuclear envelope, ER, Golgi, lysosomes, vacuoles, plasma membrane) & proteins
for secretion, Uses signal peptide to target location
Signal peptide:
20 AA at leading end of polypeptide Signal-recognition particle (SRP):determines destination
Signal-recognition particle (SRP):
brings ribosome to ER
Polyribosomes
A single mRNA can be
translated by several
ribosomes at the same
time
Mutations occur in DNA & affect RNA + Proteins
changes in the genetic material of a cell
Chromosomal Mutation
large-scale; always causes disorders or
death (eg. nondisjunction, translocation, inversions, duplications,
large deletions)
Point Mutations
change single nucleotide pair of a gene: Substitution & Frameshift (insertion/deletion)
Substitution Mutation
– replace 1 with another
Silent: same amino acid (no effect)
Missense: different amino acid (change shape & function)
Nonsense: stop codon, not amino acid
Frameshift (insertion/deletion) Mutation
mRNA read incorrectly;
nonfunctional proteins (will kill itself) (whole line - fucked)
Mutagens
substances of forces that cause mutations in DNA
Prokaryotes (everything)
Transcription & translation both in
cytoplasm
DNA/RNA in cytoplasm
RNA poly binds directly to promoter
Transcription makes mRNA (not processed)
No introns
Eukaryotes (everything)
Transcription in nucleus; translation in cytoplasm
DNA in nucleus, RNA travels in/out nucleus
RNA poly binds to TATA box & transcription factors
Transcription makes pre-mRNA -> RNA processing -> final mRNA
Exons, introns (cut out)
GENE:
A region of DNA that can be expressed to produced a final
product that is either a polypeptide or an RNA molecule
Bacterial control of gene expression (Operon)
cluster of related genes with on/off switch
Operon 3 Parts
Promoter – where RNA polymerase attaches
Operator – “on/off”, controls access of RNA poly
Genes – code for related enzymes in a pathway
Regulatory gene
produces repressor
protein that binds to operator to block RNA
polymerase
Repressible Operons
Normally ON -> OFF
Anabolic (build organic molecules)
Organic molecule product acts as corepressor → binds to repressor to activate it
Ex:. trp operon
Inducible Operons
Normally OFF -> ON
Catabolic (break down food for energy)
Repressor is active → inducer binds to &
shuts off repressor
Ex: lac operon
Gene Regulation: Negative Control
operons are switched off by active form of repressor protein
Ex: trp operon, lac operon
Gene Regulation: Positive control:
Regulatory protein interacts directly w/ genome to increase transcription
Ex: cAMP & CRP
cAMP + CRP = Positive Control
cAMP: accumulates when glucose = scarce
cAMP binds to CRP (cAMP receptor protein)
Active CRP → binds to DNA upstream of promoter, ↑ affinity of RNA polymerase to promoter, ↑ transcription
Chromatin Structure (Gene expression)
Tightly bound DNA → less accessible for transcription
DNA methylation (silence)
methyl groups added to DNA; tightly packed; ↓ transcription
Histone acetylation (power up)
acetyl groups added to histones; ;loosened;↑ transcription
Transcription Initiation
Specific transcriptionfactors (activators or repressors) bind to control elements (enhancer region)
Activators
increase transcription
Repressors
decrease transcription
Transcription Initiation Complex (Gene expression)
Activators or Repressors bind to enhancer regions + other proteins + RNA polymerase
Regulation of mRNA micro RNAs
micro RNAs (miRNAs)& small interfering RNAs (siRNAs) can bind to mRNA and degrade it (complementary bases) /block translation (less complete)
semiconservative nature of DNA
after one round of replication, every new DNA double helix would be a hybrid that consisted of one strand of old DNA bound to one strand of newly synthesized DNA
Plasmids
small circular DNA molecule found in bacteria
physically separate from chromosomal DNA & replicate independently.
Gel Electrophoresis
used to separate DNA molecules
on basis of size and charge using an electrical current (smaller = get farther thru gel)
(DNA → + pole)
Gene Transformation
bacteria takes up plasmid (w/gene of
interest)
PCR
(Polymerase Chain Reaction): amplify (copy) piece of DNA without use of cells
DNA microarray assays
study many genes at the same time
Restriction enzymes
used to cut strands of DNA at
specific locations (restriction sites)
difference in DNA replication for eukaryotes vs prokaryotes
pro: 1 origin of replication, eukaryotes = many origins
difference in cell division of eukaryotes vs prokaryotes
pro: binary fission, euk: mitosis
