Cell and Molecular Biology Exam 2

Griffith’s Experiment

Noticed that if you grow bacteria on plates, there would be two kinds, one has smooth (s strain), one has rough (strain). smooth strain made the mouse die, r strain made the mouse survive. Why does one bacterium kill, and the other doesn’t? S strain heat-killed and mouse lives. R strain with heat kill the strain, mouse dies. There was living, pathogenic s strain was recovered. The conclusion was that something from the dead s strain was taking up from the r strain, making the harmless r strain into a deadly s strain.

Avery’s Experiment

used a process of elimination with enzymes to identify DNA as the "transforming principle" that caused R-strain bacteria to become S-strain bacteria

Hershey and Chase’s Experiment

• studied the T2 phage that infects e coli. The virus sticks to the E. coli cell, and there’s genetic material inserted. Is it protein or DNA? Labeled DNA p32 and protein s35. They then let them infect coli, sheared the viral heads off of the bacteria, and then centrifuged. They then found that the infected bacteria contained p32 and not s35. This then means that the DNA was transmitted from the virus to the bacteria. DNA was determined to be the genetic factor.

Polarity (runs 5’-3')

DNA molecule has….

antiparallel strands

DNA is composed of….

A-form of DNA

• right handed

• shorter and tightly packed

B-form of DNA

• right handed

• longer and loosely packed, wider

Z-form of DNA

• left handed

• thin and long

Chromatin

Nuclear DNA + Protein

Heterochromatin

• Tightly packed DNA

• Gene Silencing

• Inactive for transcription

Euchromatin

• DNA is more unwrapped/loose

• Gene expression

• Active Transcription

“beads on a string”

What is the structure of chromatin?

Beads on a string

Partial unfolding of the chromatin fibers by gentle chemical treatments

Remodeling Complex

a multi-protein machine that uses the energy from ATP to alter nucleosomes and change the accessibility of DNA to other proteins

Nucleosomes

• Composed of 8 histones

• Disc-shaped

Packages the eukaryotic DNA into chromatin and regulates genes

What is the function of nucleosomes?

Histone tail modifications

• DNA wraps around the histone, which makes the proteins stick out.

• You can add chemical groups to certain amino acids on those tails

• Certain changes in these tails can lead to certain expressions

Lysine 9 +trimethyl

Gene silencing

Lysine 4 + trimethyl

Gene expression

Transcription

Process of copying a DNA sequence into an RNA molecule using RNA polymerase

Recognizes promotors

What does the sigma factor do in prokaryotes during transcription?W

TFIID, or TBP

What binds to the TATA box during transcription in eukaryotes?

Type of polymerase in prokaryotes

RNA polymerase

Type of Polymerase in eukaryotes

rRNA, mRNA, and tRNA

-10

Where is the TATA sequence in prokaryotes?

-25

Where is the TATA box in eukaryotes?

• 5′ cap added

• Introns spliced out

• 3′ poly-A tail added

What is mRNA processing like in eukaryotes?

Yes in prokaryotes, not in eukaryotes

Can you use mRNA right after it’s made?

Polyadenylation signals

What leads to termination in eukaryotes?

mRNA

Temporary copy of a gene used to make a protein

• made in the nucleus

• single-stranded

By RNA polymerase as a compementary strand to the DNA template

How is mRNA made?

DNA

Stores genetic information

• double stranded

• found in nucleus

Initiation (transcription)

RNA polymerase binds to the promotor region on DNA

Elongation (transcription)

RNA polymerase moves to the DNA template strand, adding complementary RNA nucleotides (A,U,C,G)

Termination (transcription)

Transcription stops when RNA polymerase reaches a termination signal on the DNA

-35 or -10

What are the promotor regions in prokaryotes?

-25,-90

What are the promotor regions in eukaryotes?

Polyadenylation signal sequence (AAUAAA)

What is the termination signal in eukaryotes?

Converting DNA to RNA sequence

Transcription creates an mRNA strand that is complementary to the template DNA strand and identical to the coding DNA strand, except RNA has U (uracil) instead of T (thymine).

Template Strand 

3’-5’

Coding strand

5’-3’

Only has exons

prokaryotes

Has introns and exons

eukaryotes

Splicing

Introns are removed and exons are joined together

Introns

noncoding regions

Exons

Coding regions

RNA Polymerase II

• Transcribes protein-coding genes (mRNA).

• Found only in eukaryotes.

• Requires transcription factors to bind DNA promoters.

CTD

• Made of repeating heptapeptides (Tyr-Ser-Pro-Thr-Ser-Pro-Ser).

• Serves as a platform for enzymes that process mRNA.

• Coordinates capping, splicing, and polyadenylation during transcription.

• Early: 5′ capping enzymes bind.

• Middle: Splicing factors attach.

• End: Poly-A tail enzymes bind.
  → Ensures transcription and mRNA processing are tightly linked.

What is CTD’s role in mRNA processing?

Nirenberg and Matthaei

• Created synthetic RNA (poly-U) → produced polyphenylalanine.

• Discovered first codon: UUU = phenylalanine.

Khorana

Made repeating RNA sequences to decode more codons

Crick/Brenner

Frameshift mutations proved that the code is triplet

Translation

• Converts mRNA → protein using ribosomes and tRNAs.

• Occurs in the cytoplasm.

Initiation (translation)

• Small ribosomal subunit binds to mRNA.

• Start codon (AUG) recognized by initiator tRNA (Met).

• Large subunit joins → complete ribosome forms.

Elongation (translation)

• tRNA enters A site → peptide bond forms.

• Chain moves to new tRNA.

• Ribosome translocates 3 bases along mRNA.

Termination (translation)

• Stop codon enters A site.

• Release factor binds → polypeptide released.

• Ribosome dissociates.

• Size: 70s (30s+50s)

• rRNAs: 16s,2 3s, 5s

What is the structure of ribosomes in prokaryotes?

• Size: 80s (40s+60s)

• rRNAs: 18s, 28s, 5.8s, 5s

What is the structure of ribosomes in eukaryotes?

They catalyze peptide bond formation and ensure there’s the correct codon-anticodon pairing.

What is the purpose of ribosomes?

Ribozyme

RNA molecule that acts as a catalyst

• can perform reactions like enzymes without using proteins

• Cloverleaf shape or L-shaped (if in 3d)

• Anti-codon loop

What is the structure of tRNA?

Messenger RNAs (mRNAs)

Code for proteins

Ribosomal RNAs (rRNAs)

Form the core of the ribosome’s structure and catalyze protein synthesis

MicroRNAs (miRNAs)

Regulate gene expression

Transfer RNAs (tRNAs)

Serve as adaptors between mRNA and amino acids during protein synthesis

Other noncoding RNAs

Used in RNA splicing, gene regulation, telomere maintenance, and many other processes

tRNA Synthetase Enzymes

How do amino acids attach to the tRNA?

It blocks binding of aminocyl-tRNA to A site of ribosome

How does Tetracycline affect translation?

It prevents the transition from initiation complex to chain elongation and also causes miscoding

How does Streptomycin affect Translation?

It blocks the peptidyl transferase reaction on ribosomes

How does Chloramphenicol affect translation?

It blocks the translocation reaction on ribosomes

How does Cycloheximide affect translation?

It blocks initiation of transcription by binding to RNA polymerase

How does Rifamycin affect Translation?

It blocks RNA polymerase in prokaryotes

How does RIfampicin affect transcription?

It binds to DNA and stops RNA elongation

How does Actinomycin D affect transcription?

Proteins degraded even when lysosomes were inactive.

How was the ubiquitin-proteasome pathway discovered?

The ubiquitin tags proteins and then the proteasome recognizes the tagged proteins and digests them

How does the 26S proteasome work?

All cells in a multicellular organism contain the same DNA, but different genes are turned on or off in each cell type

• all cells have identical DNA

• different types arise from differential gene expression

How do we know that genes specify cell fate?

Determine which genes are turned on or off in a cell

• control cell type, function and response to signals

What do transcriptional regulators do?

Structure of transcriptional regulators

DNA-Binding Domain (DBD)- binds to DNA sequences

Operon

Cluster of genes controlled by one promotor

Lactose (Lac) operon

A transcriptional repressor and activator control expression

Typtophan (Trp) operon

Single transcriptional repressor controls expression

Activators

Increase transcription by binding to enhancers or promotors

Repressors

Block transcription by binding to operator or promotors

They test regulatory DNA sequences by fusing them to a reporter gene

How do gene reporter assays work?

Histone Acetyltransferases (HATs)

Can activate gene expression by loosening chromatin

Histone Deacetyletransferases (HDACs)

Can tighten chromatin and suppress gene expression

Agouti Gene Experiment

• Controls mouse coat color & obesity.

• Under-methylated: Yellow, obese (BPA exposure).

• Hyper-methylated: Brown, healthy.

• Proves that DNA methylation can influence phenotype and be inherited.

inherited

Histone modifications can be……

Positive Feedback Loop

A transcription factor activates its own gene, maintaining its expression long-term

Cell Memory Concept

Once a gene is turned on/off, the state is inherited by daughter cells

“BAM”

• Brn2

• Ascl1

• Myt1l

What transcription factors can convert liver cells into neurons?

Genes are silenced

How do hyper-methylated genes affect?

Genes are expressed

How do under-methylated genes affect?

TBP

Recognizes TATA in promoter

TFIID

Binds TATA (through TBP), recruit TFIIB

TFIIB

Recruits Polymerase II

TFIIH

Breaks apart double helix using ATP hydrolysis and Phosphorylates (CTD)

Quantify the reporter signal (e.g., light, fluorescence) from treated samples and compare it to control samples to assess changes in reporter gene expression

• we know that only certain cells express a certain gene

How do you interpret a reporter gene assay?

• Inducible = Usually OFF, turned ON by lactose.

• Combines negative control (repressor) and positive control (CAP-cAMP).

What is the mechanism for regulation of the Lac operon?