Gene Control

Cell commitment

Even without phenotypic expression cells are committed to a particular differentiated state

DNA is ____ but _____ expressed

Unchanged, Differentially

Purpose of a Northern blot

Detects specific mRNA and is used to check the expression of a gene

• uses a probe

Southern Blot

Probe detects specific DNA.

Tandem Mass Spectrometry (How does MS/MS)

Used for peptide sequencing. Peptide is fragmented into smaller ions, and the mass differences between consecutive fragment ions reveal the amino acid sequence

RT-PCR

Synthesizes cDNA or complementary DNA from an RNA template that is amplified using PCR

Nuclear Run On

measure active gene transcription by allowing RNA polymerases in isolated nuclei to extend nascent RNA strands with labeled nucleotides. The labeled RNA is then hybridized to gene-specific probes to identify which genes were being transcribed at the time of isolation.

DNA microarray

used to compare gene expression under different conditions

ex. comparisons of gene expressions in diseases vs normal cell 

miRNA

• Regulates gene expression post-transcriptionally by binding to complementary sequences on mRNAs, leading to degradation or translational repression.

• single stranded RNA

  • 20-25 base pairs

siRNA

• double stranded

• Defends against foreign nucleic acids (like viruses, transposons) by guiding the cleavage and degradation of target mRNAs.

• Silences transposable elements in germ cells and helps maintain genome stability.

• 21-31 double strands

lncRNA

• 200 bp long regulatory RNA

• bidirectional promoter

• Regulates gene expression at multiple levels:

  • chromatin modification → repression

  • transcription:

    • competes for transcription regulatory proteins → repression

    • recruit transcription activator → activator

    • post-transcriptional inhibition

• Xist lncRNA → coats one X chromosome in females to inactivate it (X-chromosome inactivation).

Steps of RNA Transcription

Initiation

Elongation

Termination

Promoter

Where RNA polymerase first binds

Important Sequences of the Promoter

• -35 and -10

  • aka. consensus sequence

-35 = TTGACA

-10 = TATAAT

What is needed for RNA Polymerase to recognize the -35 and -10 sequences?

sigma factor

Transcriptional start site

+1

Elongation (transcription)

The chaining of RNA Nucleotides together

• rNTP enter and start chaining together

• nucleotide is added to the 3’ end of the RNA

Kinetic Proofreading

Involves pyrophosphate lysing the bond or something of the RNA

Nucleolytic proofreading

involves nuclease within RNA polymerase to lyse the bond of the RNA

Rho Dependent Termination (transcription)

• A termination mechanism that requires the Rho protein, a helicase.

• Rho binds to a rut site (Rho utilization site) on the nascent RNA.

• It moves along the RNA (using ATP) toward the RNA polymerase.

• When RNA polymerase pauses at a termination sequence, Rho catches up.

• Rho unwinds the RNA-DNA hybrid inside the transcription bubble, releasing the RNA transcript.

Rho-Independent Termination (Transcription)

• The RNA transcript forms a GC-rich hairpin loop followed by a series of uracils (poly-U tail).

• The hairpin causes RNA polymerase to pause.

• The weak A–U base pairing between RNA and DNA makes the RNA-DNA hybrid unstable.

• The RNA transcript dissociates, ending transcription.

tRNA

the link between the mRNA and the polypeptide

is in a three leafed clover shape

tRNA synthetase

add amino acids to the tRNA by an active site.

charges the tRNA

amino acids are attached by their carboxyl group to the 3’OH of the tRNA

Structure of Ribosomes

2/3 RNA, 1/3 protein

Contains a large ribosomal subunit and a small ribosomal subunit

Ribozyme

all enzymatic activity in the ribosome is by the RNA components = ribozyme

Binding sites of Ribosomes

A site

P site

E site

A site of Ribosome

aminoacyl site, binds an aminoacyl-tRNA

P site of ribosome

peptidyl site, binds a tRNA with an attached growing polypeptide chain

E site of ribosome

exit site, binds to deacylated tRNA that is to be released from ribosome

Translation Initiation

1. Small ribosomal subunit with translation initiation factor bind and then bind to mRNA

2. small ribosomal subunit with bound initiation tRNA moves along mRNA and searches for the first AUG

3. large ribosomal subunit binds

4. Charged tRNA binds to A site

5. firs peptide bond forms

need for gene regulation in bacteria

• Control gene expression in response to environmental changes.

• Energy conservation: Proteins made only when needed.

• Types of genes:

  • Constitutive → always on (basic survival).

  • Regulated → active only under certain conditions.

• Regulation types:

  • Temporal (right time)

  • Spatial (right place)

  • Quantitative (right amount)

Cis elements

• DNA sequences located on the same molecule of DNA as the gene(s) they regulate.

• Examples: Promoter, Operator, Enhancer/Silencer sequences.

Trans elements

• Regulatory molecules (usually proteins or RNAs) that diffuse through the cell and act on cis elements.

• Can regulate genes on other DNA

  • Examples: Repressor protein, Activator protein, Sigma factors.

Ara operon in the absence of arabinose

the araC protein is made as usual but binds to each other to form a dimer and blocks the RNA polymerase from transcribing the genes

ara operon in the presence of arabinose

arabinose binds to the araC proteins so they loosen their grips on each other and move to adjacent areas near the promoter. Then CAP protein binds to the CAP site in the absence of glucose and activates transcription.

CAP protein in lac operon

enhances transcription. Needs cAMP to be activated and bind to the cAMP site.

In the presence of glucose, the CAP protein is INACTIVATED

best conditions in a lac operon is for lactose to be present but no glucose.

gene regulation by mRNA

Gene regulation at the mRNA level happens after transcription but before (or during) translation. It controls how much protein is made from the mRNA.

Riboswitch

RNA molecule acting as a switch depending on the effector molecule binding

main regulation in eukaryotes

🔴 Negative Regulation (Repressor-based)

• Similar to bacterial negative inducible systems (e.g., lac operon).

• Repressor proteins bind promoters → block transcription.

• A signal (e.g., heat shock, stress) inactivates the repressor → gene is expressed.

🟢 Positive Regulation (Activator-based)

• Many eukaryotic genes are default OFF.

• Require activator proteins to initiate transcription.

• Activators may:

  • Be tissue-specific.

  • Require cofactors (e.g., steroid hormones).

• Common in higher organisms → ensures genes expressed only in correct tissue, time, or signal.

Summary:

• Negative regulation = block lifted → gene ON.

• Positive regulation = activator required → gene ON.

Front (Q): What is chromatin remodeling?

• The process of altering nucleosome structure/position to regulate DNA accessibility.

• Involves ATP-dependent remodeling complexes + histone modifications.

  • Switches chromatin between euchromatin (open/active) and heterochromatin (closed/silent).

What are the main mechanisms of chromatin remodeling?

• Nucleosome repositioning/sliding → DNA exposed for transcription factors.

• Histone modifications:

  • Acetylation → loosens chromatin → transcription ON.

  • Deacetylation → tightens chromatin → transcription OFF.

    • Methylation → context-dependent (ON or OFF).

    • Phosphorylation, ubiquitination: additional regulation layers.

Phosphorylation, acetylation:

→ add negative charges

→ neutralize basic histone

→ repel DNA or other modified histone

→ interaction with regulatory protein

micrococcal nuclease

Micrococcal nuclease digests exposed DNA between nucleosomes. Then, only purify the ones that have histones attached to it, getting rid of the histone protein parts and the DNA left will be sequenced.

 Break in DNA means a histone was present in tumor vs normal cell

So, if you map all the cut sites across the genome, you can figure out nucleosome positioning:

• Break in DNA sequence after MNase digestion = accessible, unprotected DNA (between nucleosomes).

• No break (protected region) = nucleosome present.

3C, 5C experiment

Involves immunoprecipitation using chromatin

LCR

• controls expression of all the globin genes

• functions to keep open chromatin conformation

• deletion leads to low globin gene expression - Hispanic thalassemia