LESSON 5 PROTEIN SYNTHESIS Flashcards
Different from protein synthesis
DNA REPLICATION
DNA copies itself on the process of conserving it
DNA REPLICATION
Replication fork (two strands) is separated by helicase
DNA REPLICATION
Helicase and isomerase – enzymes absent in PCR
DNA REPLICATION
photocopy/replication of DNA outside the body (in vitro)
PCR
Principle is based on the process of DNA replication
PCR
Number of copies can be as high as 10^9
PCR
DNA polymerase from humans: happens at 37oC
DNA REPLICATION
Taq polymerase from thermophilus bacteria: can withstand high temperature (varying from 90 to 72 to 55 oC)
PCR
Separation of two strands thru helicase
DNA REPLICATION
Separation of two strands thru heat at 90oC (denaturation)
PCR
Primers are made in the body
DNA REPLICATION
Primers are commercially available
PCR
DNA template is produced by the body
DNA REPLICATION
DNA template is extracted (ex. RNA from covid is converted to cDNA)
PCR
Makes similar copies of the same DNA called
DNA template
– construction worker of daughter strands; builds and adds nucleotides
Uses DNA polymerase or Taq polymerase)
– acts as bookmarks, markers, or flags to tell which one to copy
Primers
can be reagents made at manufacturing
Primers
used for cutting or copying DNA parts
Primers
Place for DNA polymerase to attach to DNA strand Feature in humans
RNA primers
Place for DNA polymerase to attach to DNA strand Feature in PCR
DNA primers
Separates the two strands of DNA Feature in humans
Helicase
Separates the two strands of DNA Feature in PCR
Heat
Name of enzyme that elongates new strand of DNA Feature in humans
DNA polymerase
Name of enzyme that elongates new strand of DNA Feature in PCR
Taq or other thermophillic DNA polymerase
What the primers are made out of (DNA or RNA?) Feature in humans
RNA
What the primers are made out of (DNA or RNA?) Feature in PCR
DNA
Items that are common in both situations that has not been mentioned
Feature in humans
Feature in PCR
A method widely used in molecular biology to make many copies of a specific DNA segment
DNA REPLICATION
The biological process of producing two identical replicas of DNA from one original DNA molecule
PCR
An in vitro process, which occur inside a test tube
DNA REPLICATION
An in vivo process, which occur inside living cells
PCR
Main goal is to produce exponential number of copies of a single DNA fragment
DNA REPLICATION
Main goal is to copy the whole genome at once
PCR
The target is shorter
DNA REPLICATION
The target is longer
PCR
A discontinuous process, which proceeds through 30-40 cycles
DNA REPLICATION
A continuous process
PCR
DNA duplex is opened up by the enzyme ATP-dependent helicase
PCR
Uses DNA primers
DNA REPLICATION
Uses RNA primers synthesized by primase
PCR
Uses thermophilic DNA polymerase such as Taq DNA
DNA REPLICATION
Uses DNA polymerase
PCR
Taq polymerase is not featurerich and also, it has no proofreading ability
DNA REPLICATION
DNA polymerase is contained high fidelity, speed, proofreading and repair
PCR
No replication fork forms
DNA REPLICATION
Replication fork forms
PCR
Taq polymerase does not contain the 5’ to 3’ exonuclease activity
DNA REPLICATION
DNA polymerase has the 5’ to 3’ exonuclease activity to degrade RNA primers
PCR
Taq polymerase operates at high temperatures such as 72 °C
DNA REPLICATION
DNA polymerase operates at physiological temperature, which is 37 °C
PCR
Serves as a simple approach for in vitro DNA synthesis
DNA REPLICATION
A complex process, which depends upon a well defined but complex set of enzymes and cofactors
PCR
Speed of Synthesis: 1-4 kb/min (faster)
DNA REPLICATION
Speed of Synthesis: 1 kb/s
PCR
Error Rate: 1 in 9000 bases (less error)
DNA REPLICATION
Error Rate: 1 in 100,000 bases
PCR
DNA duplex is melted by using heat, which is >90 °C
DNA REPLICATION
– template or code and basis; enclosed only in the nucleus
DNA
– copying of DNA
Transcription
- copying a portion of DNA
Translation
Translation end product is
mRNA
: tough and can withstand oxidative stress in the cytoplasm unlike DNA
RNA
: can go outside the nucleus unlike DNA
mRNA
what has been copied will be translated and interpreted to become an amino acid to protein
mRNA
can go to the cytoplasm then to the ribosome (factory)
mRNA
: area for translation
Ribosome
the cellular process by which DNA is copied to RNA
Transcription
occurs in the nucleus
Transcription
process by which RNA transcripts are turned into proteins and peptides
Translation
occurs in the cell cytoplasm
Translation
Transcription Three steps:
Initiation: when does it start?
Elongation: how does it extend?
Termination: when does it end?
: when does it start?
Initiation
: how does it extend?
Elongation
: when does it end?
Termination
Begins once the promoter gene sequence is detected by the
transcription factors called TATA box
Initiation
transcription factors called
TATA box
Has thymine and adenine sequences
TATA box
Recruits the transcription factors, mediator proteins,
and RNA polymerase
TATA box
: add nucleotides and important forcreating mRNA
RNA polymerase
A portion of DNA is already transcripted to be an mRNA
Initiation
is a DNA sequence that indicates which specifies to other molecules where transcription begins.
TATA box
• TATA box + transcription factors (RNA polymerase)=
TRANSCRIPTION INITIATION COMPLEX
Site where mRNA will be synthesized
TRANSCRIPTION INITIATION COMPLEX
Aka initiation bubble
TRANSCRIPTION INITIATION COMPLEX
unwound DNA strand
Elongation
RNA polymerase
Add the complementary nucleotides to builds the mRNA molecule, using complementary base pairs.
Elongation
RNA polymerase
Very important for elongation
RNA polymerase
Building of mRNA to be ready for interpretation as amino acid
Elongation
C
G
T
A
G
C
A
U
Two strands of DNA are separated
1) Template strand, 2) Coding strand
Elongation
: is template used to make mRNA (5’ to 3’ strand) (active in transcription)
Template strand (3’ to 5’)
: mRNA Like strand except for the Uracil of course (no role in transcription）- fast
Coding strand (5’ to 3’)
Opposite in replication; requires both lagging strand and
leading strand; only requires template strand
elongation
The RNA transcript will undergo processing through the addition of a modified guanine nucleotide called 5’ cap or G cap at the 5’ end and 50 - 250 adenine nucleotide called poly-A tail at the 3’ end.
Termination
RNA polymerase crosses a stop (termination) sequence in the gene. The strand is called a [?]
pre-mRNA strand
pre-mRNA must be processed to a
“mature mRNA”
Why process pre-mRNA further?
1. Check for [?]
2. Allows the mRNA molecule to be exported to the [?]
3. Additional protection from [?] and gate pass [?]
4. Removes [?]
mistakes or errors
ribosomes
photochemical mutations ; G cap & Poly-A tail
introns (non coding regions)
Can go out of the nucleus once checked
mRNA
Poly-A tail: many adenine
5’ guanine nucleotide cap
gate pass G cap & Poly-A tail
: process of removing introns (light orange)
a. spliceosome
: hard bound
G cap & Poly-A tail
: blank pages
Introns
mRNA leaves the nucleus through its pores and goes to the ribosomes
mRNA Transcript
mRNA enters the ribosome
Translation
: pin-like; reads anticodons
transfer RNA (tRNA)
: 3 consecutive nucleotides
codons
Decoding of mRNA
Translation
: instructions in mRNA in groups of 3 nucleotides
Codons
a. different codons for aminoacids
61
b.: start codon to begin translation
AUG
c.: finished polypeptide
Stop codons
tRNA reads the mRNA from the 5’ to 3’ end
Translation
tRNA has an anti-codon that binds to matching mRNA through base pairing
Translation
tRNAs enter slots/ sites in the ribosome and bind to codons
Translation
: mRNA site
small subunit
: tRNA site (flashlight-like with anticodons below)
large subunit
: accepts the incoming aminoacylated tRNA
- A site (amino-acyl)
aka “landing site”
- A site (amino-acyl)
: holds the tRNA which is linked to the growing polypeptide chain
- P site (peptidyl)
: holds the tRNA before it leaves the ribosome
- E site (exit)
very potent antibiotics
Azithromycin, Gentamicin, Chloramphenicol, Erythromycin
principle: stops translation/protein synthesis in bacteria; attacks prokaryotic ribosome
Azithromycin, Gentamicin, Chloramphenicol, Erythromycin
Free ribosomes in prokaryotes
PROKARYOTIC RIBOSOMES
Large ribosomes that facilitate translation in eukaryotes
EUKARYOTIC RIBOSOMES
Found inside bacteria and archaea
PROKARYOTIC RIBOSOMES
Small and mass is 27000 kd
PROKARYOTIC RIBOSOMES
Sedimentation coefficient is 70S
PROKARYOTIC RIBOSOMES
Diameter is ~200 A
PROKARYOTIC RIBOSOMES
Made up of 50S and 30S subunits
PROKARYOTIC RIBOSOMES
Large subunit is made up of two rRNA molecules: 23S rRNA and 5S rRNA
PROKARYOTIC RIBOSOMES
Made up of 60% rRNA and 40% ribosomal proteins
PROKARYOTIC RIBOSOMES
Occur free in the cytoplasm
PROKARYOTIC RIBOSOMES
Found in animals, plants, fungi, and other unicellular eukaryotes with a nucleus
EUKARYOTIC RIBOSOMES
Large and mass is 42000 kd
EUKARYOTIC RIBOSOMES
Sedimentation coefficient is 80S
EUKARYOTIC RIBOSOMES
Diameter is ~250-300 A
EUKARYOTIC RIBOSOMES
Made up of 60S and 40S subunits
EUKARYOTIC RIBOSOMES
Large subunit is made up of three rRNA molecules: 28S rRNA, 5.35 rRNA, & 5S rRNA
EUKARYOTIC RIBOSOMES
Made up of 40% rRNA and 60% ribosomal proteins
EUKARYOTIC RIBOSOMES
Most are attached to the outer surface of nucleus and endoplasmic reticulum
EUKARYOTIC RIBOSOMES
Amikacin
Aminoglycosides
Dibekacin
Aminoglycosides
Gentamicin
Aminoglycosides
Kanamycin
Aminoglycosides
Neomycins
Aminoglycosides
Streptomycin
Aminoglycosides
Tobramycin
Aminoglycosides
Chloramphenicol
Amphenicols
Thiamphenicol
Amphenicols
Azithromycin
Macrolides
Carbomycin A
Macrolides
Clarithromycin
Macrolides
Erythromycin
Macrolides
Eperezolid
Oxazolidinones
Linezolid
Oxazolidinones
Posizolid
Oxazolidinones
Radezolid
Oxazolidinones
Sutezolid
Oxazolidinones
Pristinamycin
Streptogramins
Quinupristin
Streptogramins
dalfopristin
Streptogramins
Virginiamycin
Streptogramins
Doxycycline
Tetracyclines
Chlortetracycline
Tetracyclines
Lymecycline
Tetracyclines
Meclocycline
Tetracyclines
Minocycline
Tetracyclines
Peptide elongation at the bacterial 30S ribosomal subunit
Aminoglycosides
Protein elongation by overlapping with the binding site at the A-site of 50S ribosomal subunit
Amphenicols
Peptide-bond formation and ribosomal translocation
Macrolides
Peptide-bond formation by blocking tRNA binding at the A-site of 50S ribosome
Oxazolidinones
Protein elongation at the A- and P-sites of 50S ribosome
Streptogramins
Polypeptide synthesis by sterically blocking the recruitment of the aminoacyl-tRNA at the A-site of the bacterial 30S ribosomal subunit
Tetracyclines
Kidney injury
Aminoglycosides
vestibular
Aminoglycosides
Aplastic anemia
Amphenicols
bone marrow suppression
Amphenicols
neurotoxicity
Amphenicols
Myopathy
Macrolides
QT prolongation nausea
Macrolides
Nausea
Oxazolidinones
Streptogramins
bone marrow suppression
Oxazolidinones
lactic acidosis
Oxazolidinones
myalgia
Streptogramins
arthralgia
Streptogramins
Phototoxicity
Tetracyclines
secondary intracranial hypertension
Tetracyclines
teeth discoloration, steatosis
Tetracyclines
liver toxicity
Tetracyclines
TAC
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
AUG
UAC
MET
TGA
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
ACU
UGA
THR
TCG
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
AGC
UCG
SER
ACC
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
UGG
ACC
TRP
TTC
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
AAG
UUC
LYS
GAT]
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
CUA
GAU
LEU
TAG
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
AUC
UAG
ILE
ATG
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
UAC
AUG
TYR
AGG
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
CGT
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
CTG
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
AAG
TRANSCRIPTION (mRNA):
TRANSLATION (tRNA):
Amino acid:
