Molecular Genetics Exam 4

Crick

proposed tRNA as the adaptor molecule in protein synthesis.

discovery of tRNA

Attached radioactive amino acid to tRNA

radioactive amino acid is transferred from tRNA to the growing polypeptide chain.

proved that tRNA was the key molecule in translating codons into amino acids.

tRNA specificity

mRNA codon matches anticodon on tRNA ensuring the correct amino acid is added to the polypeptide chain.

Nitrocellulose binding assay

nitrocellulose filter binds protein but not RNA, so tRNA bound to ribosome are isolated

Ribosome + short-predetermined mRNA + radioactive amino acids are used to identify which amino acids are incorporated into the protein.

supressor tRNA

A type of tRNA that can recognize and insert an amino acid at a stop codon, allowing for the read-through of the codon during protein synthesis.

example - changing anticodon from AUG 5’ → AUC 5’ to bind to stop codon and add an an amino acid anyway

wobble rules

1. first 2 bp correspond with tRNa anticodon

2. 1st base of the anticodon defines specificity

   1. A/C = more specific

   2. U/G = less specific

The wobble rules explain how the third base pairing between tRNA anticodons and mRNA codons can be flexible, allowing for some variation in base pairing. This flexibility enables one tRNA to recognize multiple codons that code for the same amino acid, thus optimizing protein synthesis.

tRNA aminoacylation

The process of attaching an amino acid to its respective tRNA molecule, catalyzed by aminoacyl-tRNA synthetases. This activation is essential for accurate translation of the genetic code into proteins. reversible

rossman fold

common structural motif of class 1 catylitic domains for tRNA aminoacylation with a distinct beta-alpha-beta structure that facilitates the binding of tRNA and amino acids.

class 1 aminoacyl-tRNA synthetases

add amino acids to 2’ OH of the ribose of the adenine in the tRNA's acceptor stem, crucial for protein synthesis.

usually undergoes transesterification that transfers the amino acid to the 3’ oh

class 2 aminoacyl-tRNA synthetases

add amino acids to 3’ OH of the ribose of the adenine in the tRNA's acceptor stem, crucial for protein synthesis.

rodin-ohno hypothesis

a theory proposing that the evolution of aminoacyl-tRNA synthetases is influenced by the co-evolution of tRNA and their corresponding amino acids.

suggests that the two distinct classes of these enzymes—Class I and Class II—originated from opposite strands of the same ancestral gene.

t-box riboswitch evolution

enzyme that can recognize tRNA aminoacylation status and plays a part of a regulatory feedback ensures that genes like tRNA synthetases are upregulated when uncharged tRNA accumulates—i.e., when there is a need for more aminoacylation activity.

tyrS

gene that encodes the tRNA synthetase for tryptophan, playing a critical role in amino acid incorporation during protein synthesis.

expressed at low levels of tyr

tRNA aminoacylation proofreading

because the codon arm and the anticodon arm are chemically distinguishable, and tRNA synthetases have a proofreading function via a secondary active site that hydrolizes incorrect amino acids to ensure fidelity in protein synthesis by correcting errors in aminoacylation.

small subunit (30s/40s)

interacts with mRNA

facilitates mRNA-tRNA binding in decoding center

large subunit (50s/60s)

promotes peptide bond formation in peptidyl transferase center GTP activation center that drives translation elongation.

translation initiation

matches mRNA with ribosome

assemble translation competant ribosome

start codon binds to initiator tRNA

polysome

a complex of multiple ribosomes translating a single mRNA strand simultaneously, enhancing protein synthesis efficiency.

16S rRNA

the rRNA component of the 30S subunit

5S and 23S rRNA

the rRNA components of the 50S subunit in prokaryotic ribosomes, playing crucial roles in the structure and function of the ribosome.

ribozyme

a type of RNA molecule that can catalyze biochemical reactions, similar to protein enzymes.

ribosome is a ribozyme

without rRNA (mutated sequence) ribosome doesnt function

without protein, ribosome can still conduct peptidyl transferase activity due to the rRNA component, emphasizing the role of RNA in catalysis.

A site

the location on the ribosome where aminoacyl-tRNA binds during protein synthesis, essential for the elongation phase of translation.

P site

the location on the ribosome where the peptidyl-tRNA resides during protein synthesis, playing a crucial role in peptide bond formation.

E site

the exit site on the ribosome where tRNA, after donating its amino acid, leaves the ribosome during protein synthesis.

puromycin

antibiotic

halts translation by occupying the A-site as a tRNA mimic and prevents further elongation

shine-dalgarno sequence

A ribosomal binding site in bacterial mRNA (6-11 bp upstream of start codon) that helps align the mRNA on the ribosome for translation initiation by binding to the 16S rRNA of the 30S subunit

RPS1

A component of the ribosomal 30S subunit in bacteria, playing a role in mRNA binding and translation initiation.

Kozak sequence

A specific sequence of nucleotides surrounding the start codon in eukaryotic mRNA that enhances the efficiency of translation initiation.

fMet-tRNA

The initiator transfer RNA in prokaryotes that carries formylmethionine, playing a crucial role in the initiation of protein synthesis.

IF1

blocks A site of 30S subunit

IF2

recruits initiator trNA to P-site

hydrolized and released at binding of large subunit (50S)

IF3

prevents premature joining of the 50S subunit to the 30S subunit during initiation of translation.

EF-Tu

most abundant protein in E.coli that delivers aminoacyl-tRNA to the ribosome during protein synthesis.

GTP hydrolysis releases proteinn and incorporates tRNA

EF-Ts

a translational elongation factor that regenerates active EF-Tu by catalyzing the exchange of GDP for GTP.

nucleotide exchange factor

EF-G

facilitates translocation of ribosome along mRNA

binds to large subunit A-site (displaces peptidyl-tRNA)

on binding, GTP hydrolyzes and protein changes conformation to bind to the small subunit A-site

mimics EF-Tu

accomodation

the process by which the aminoacyl-tRNA anticodon matches the mRNA codon in the ribosome's A-site, ensuring correct translation and peptide bond formation.

corect interactions rotate the tRNA such that its amino acid is positioned for peptide bond formation.

RF-1

a release factor that recognizes stop codon UAG during translation termination, promoting the hydrolysis of the peptidyl-tRNA in the P-site.

RF-2

a release factor that recognizes the stop codon UGA and UAA during translation termination, facilitating the release of the polypeptide from the ribosome.

class 1 RF

A type of release factor that recognizes specific stop codons during translation termination to promote polypeptide release.

class 2 RF

responsible for release of class 1 RF

RF-3

binds to GDP on the ribosome to stimulate the release of class 1 RF (weak bond if class 1 RFs arent there)

peptide release oxidizes GDP to GTP

GTP hydrolized to GDP, releasing the protein

RRF

binds to A-site

recruits EF-G

EF-G catalyzes translocation and dissociation of ribosomal subunits

IF-3 binds to small subunit to prevent reassociation

best antibiotic trait

inhibiting elongation, because preventing translation at any position during elongation prevents protein production

ex. chloramphenicol inhibits peptidyl transfer

eIF1A

IF1 functional homolog

binds to A-site to prevent initiator tRNA binding

eIF1

IF3 functional homolog

sits next to E-site

prevents tRNA binding

prevents large subunit binding

eIF4F

protein complex that brings mRNA to small subunit in eukaryotes

eIF4E

binds to 5’ cap of mRNA

eIF4G

binds eIF4e and eIF3 to link mRNA and 43S subunit

eIF4A

RNA helicase (ATPase)

works with eIF4B to find start codon

eIF4B

stabilizes eIF4A and helps in scanning for start codon

eIF3

helps eIF4F recruit mRNA by binding to eIF4G

eIF5

a protein that promotes the assembly of the ribosomal pre-initiation complex and facilitates the release of eIF2-GDP.

eIF5B

homolog of IF2

catalyzes dissociation of initiation factors from 48s complex and allows for binding of large subunit (60S)

closed loop model

eIFG (bound to eIFE, bound to 5’ cap of mRNA) binds to PABP on 3’ poly-A tail of mRNA, creating a loop that enhances translation efficiency and stability of mRNA.

evolutionary advantage of 3” UTR

ribosomal machinery doesnt need to scan through 5’ UTR to find start codon; thus, translation is initiated more efficiently and quickly, allowing for better regulation of gene expression.

cap-dependent translation

A mechanism of protein synthesis in which the ribosome recognizes and initiates translation at the 5' cap structure of the mRNA. This process is crucial for efficient translation and regulation of gene expression.

occurs 99% of the time

IRES

allow ribosomes to initiation of translation in the middle of mRNA strands or on mRNA strands with no 5’ cap

cap-independent translation (1% of the time)

if eIF4F is destroyed

host mRNA cannot be translated, but mRNA with IRESes can be translated

a way viruses can inhibit host translation without affecting their own

RNAse-L

non-specific RNAse that degrades all RNA it encounters

expressed during infection to limit viral replication and spread.

tmRNA

recruited when mRNA is damaged or does not have a stop codon

mix of tRNA and mRNA

recruited by EF-G to empty A-site

deposits alanine and fills empty mRNA spot

codes for a tagging sequence, indicating that the protein be degraded

has proper stop codon and rescues stalled ribosomes during translation termination.

TAP-tag approach

A method used to purify and identify proteins and their interactions by tagging them with a peptide that can be selectively captured and eluted from complexes.

Calmodilin binding peptide

binds to target protein

programmable

TEV protease cleavage site

a specific amino acid sequence recognized by TEV protease, allowing for precise cleavage of proteins

cleaves target protein from protein A

Protein A

a bacterial protein that binds immunoglobulin G (IgG) and is commonly used in protein purification.

IgG

an antibody that binds to specific antigens (Protein A), aiding in the isolation of proteins

GST-tag

a protein tag derived from glutathione S-transferase that facilitates protein purification and detection.

the gene for GST is inserted next to the gene for the protein of interest

isolated through GST sensitive antibody

ddNTP

a type of nucleotide used in Sanger sequencing that terminates DNA strand elongation because the sugar lacks an OH group to continue synthesis

di-terminator nucleotide

Nucleotide that halts DNA synthesis during sequencing due to missing 3'-OH group and marks end with flourescent label.

allows for machine sequencing through capillary column

only works for short reads

pyrosequencing

A sequencing technique that uses light emission to detect nucleotide incorporation in real-time as DNA is synthesized, allowing for rapid and accurate sequencing.

can be read by machine with chip (contains thousands of sequences)

only works for short sequences

illumina sequencing

combines di-terminator sequencing and pyrosequencing to produce millions of sequences simultaneously. It uses reversible terminator chemistry and generates highly accurate short reads.

limitations to viral gm

only applies to small subset of diseases (caused by 1 or few genes)

the location of integration into genome is not guaranteed anc can encorporate into a detrimental location

viral uptake of human DNA

modify human DNA to carry viral “packaging signal” to facilitate the incorporation of viral genomes into host cells during infection, enabling the virus to replicate within the human DNA.

ZFN

Zinc Finger Nucleases, a type of engineered endonuclease that facilitates targeted gene editing by inducing double-strand breaks in specific DNA sequences.

problem: they can cause off-target effects, leading to unintended mutations in the genome.

TALEN

Transcription Activator-Like Effector Nucleases, a type of engineered protein used for targeted gene editing by creating double-strand breaks at specific DNA sites, offering higher precision than traditional methods.

problem: they may also result in off-target effects, potentially causing undesired changes in the genome. takes too long

CRISPR/Cas9

a revolutionary gene-editing technology that allows for precise modifications in DNA by utilizing a guide RNA to direct the Cas9 nuclease to specific genomic locations, enabling targeted gene disruption or insertion.

better than previous methods: it is faster, cheaper, and more specific

Cas9

endonuclease that performs the actual cutting of DNA during the CRISPR/Cas9 gene-editing process, guided by RNA to the targeted sequence.

gRNA

a short synthetic RNA molecule that guides the Cas9 nuclease to the specific DNA sequence to be edited in the CRISPR/Cas9 system.

tracrRNA

a component of the CRISPR/Cas9 system that forms a complex with gRNA and Cas9, helping to provide the necessary structure for targeting specific DNA sequences.