Unit 6: Gene Expression and Regulation

Thomas Hunt Morgan

Worked with fruit flies

Did not know if the DNA or protein that make up chromosomes are the genes

What did Thomas Hunt Morgan conclude?

Genes are on chromosomes

Frederick Griffith

Studied bacteria to find a cure for pneumonia

What did Griffin discover?

• Harmless live bacteria mixed with heat-killed infectious bacteria caused diseases in mice

• Substance passed from dead bacteria to live bacteria - “transforming factor”

Avery, McCarty, & MacLeod

Purified both DNA and proteins from bacteria

Avery, McCarty, & MacLeod, what results did they get?

• Injected protein into bacteria → no effect

• Injected DNA into bacteria → transformed harmless bacteria into virulent bacteria

Hershey and Chase

Used bacteriophages (viruses that infect bacteria) with radioactive sulfur and phosphorous

Hershey and Chase, results from sulfer and phosphorous.

• Radioactive sulfur did not enter the bacteria

• Radioactive phosphorus did enter the bacteria → DNA is “transforming factor”

Chargaff Discovery

DNA composition varies from species to species but the bases are present in a characteristic ratio

In humans, what are the DNA bases? What are their percentages?

A = 30.9%

T = 29.4%

G = 19.9%

C = 19.8%

Watson and Crick

• Developed the double helix model of DNA

• Used a photograph taken by Rosalind Franklin

Pyramidines

Nitrogen bases with 1 ring (T & C)

Purines

Nitrogenous bases with 2 ring (A & G)

How do the DNA strands run?”

The strands are antiparallel

Which side on a strand has a phosphate and which doesn’t.

• 3’ = no phosphate on end

• 5’ = phosphate on end

What holds the strands together and how many of this thing? Which bases go together?

• Hydrogen bonds hold the two strands together

  • 2 between A & T

  • 3 between G & C

What holds the phosphates and sugars together?

Phosphodiester bonds

DNA in Prokaryotes vs. Eukaryotes

DNA Replication

Base pairing allows each strand to serve as a template for a new strand

How is DNA replication a semi-conservative process?

Each double helix consists of a parent/template strand and a new DNA strand

When does DNA replication occur?

S phase of the cell cycle

Enzymes involved in DNA Replication

Helicase, Topoisomerase, DNA polymerase, RNA polymerase (primase), Ligase

Helicase

unwinds part of the DNA double helix

Topoisomerase

helps relieve the strain of unwinding by breaking, swiveling, and rejoining DNA strands

DNA polymerase

Connects nucleotides together to make a strand; proofreads and edits as it builds the new strand.

There are multiple DNA polymerases;

RNA polymerase (primase)

adds a few nucleotides of RNA to get the process started

Ligase

connects DNA fragments together

How does DNA replication start?

First DNA helicase unwinds the DNA strands.

Topoisomerase relaxes supercoiling in front of the replication fork.

How does DNA replication continue?

Then complementary nucleotides match with the nucleotides on the original DNA molecule; the new nucleotides are connected together to make a strand

What is the difference between DNA and RNA polymerase?

• DNA polymerase connects the nucleotides but can only add nucleotides to a 3’ end of a growing DNA strand

• RNA polymerase (primase) adds a few nucleotides so DNA polymerase can get started; the RNA nucleotides are later replaced

Leading strand

Made continuously in the 5’ to 3’ direction

Lagging strand

Made in Okazaki fragments that are later joined together by ligase

Telomeres

The ends of chromosomes in eukaryotes that are repeating, non-coding sequences that serve as protective caps

Why is there an aging process in eukaryotes?

Chromosomes get shorter with each replication (limits # of cell divisions)

Telomerase

Can add DNA bases at 5’ end; high activity in stem cells and cancers but not in most somatic cells

Do prokareyotes have telomeres?

No since their chromosomes are circular

Dna Replication in Prokaryotes vs. Eukaryotes

RNA

• Ribose sugar

• Uracil instead of thymine

• Single stranded

Types of RNA

mRNA, tRNA, rRNA, and microRNA

Difference in DNA and RNA

What determines RNA function?

The sequence of the RNA bases, together with the structure of the RNA molecule

mRNA

Carries information from DNA to the ribosome

tRNA

Carries amino acids to the ribosome

rRNA

Building blocks of ribosomes

microRNA

Small RNA molecules that bind to other RNA molecules to degrade them

Transcription

The nucleotide sequence in the DNA is used to make a complementary sequence in mRNA 

What enzymes are used in transcription?

Uses many of the same enzymes from DNA replication - helicase, topoisomerase, RNA polymerase, etc.

How does transcription happen?

RNA polymerase uses a single strand of DNA to make mRNA; works in the 5’ to 3’ direction

It makes a mRNA strand identical to the coding DNA strand (but swap U for T).

Which DNA strand is used in transcription?

The DNA strand that is used is called the template strand, noncoding strand, minus strand, or antisense strand.

Are DNA and mRNA strands different?

Yes because the strands of DNA are complementary and antiparallel, the mRNA made from the strands are not the same → code for different proteins

How do the bases change in DNA and RNA?

DNA RNA

A → U

T → A

G → C

C → G

Post-Transcriptional Modification

Before the mRNA leaves the nucleus - 

• A poly-A tail is added

• A GTP cap is added

• Splicing by spliceosomes

Introns

Stay in the nucleus; do not code for proteins

Exons

Exit the nucleus to go to the ribosome; do code for proteins

Alternative splicing

Different versions of the mRNA result from combining different exons

Where does post-transcirptional modification occur?

In eukaryotes only

Translation

Three steps - initiation, elongation, and termination

Where does translation occur?

• Occurs at ribosomes

  • Free ribosomes in prokaryotes

  • Free ribosomes or bound ribosomes (to rough ER) in eukaryotes

What is the difference between when transcription occurs in prokaryotes vs eukaryotes

• In prokaryotes, translation occurs as the mRNA is being transcribed

• In eukaryotes it occurs after transcription 

Initiation

• Small ribosomal subunit binds to mRNA and an initiator tRNA

• Then the large ribosomal subunit attaches

Elongation

• For each codon, a tRNA with a corresponding anticodon brings an amino acid to the ribosome

• The amino acid is added to the preceding one by a peptide bond

Codon

3 bases on the mRNA

In what direction does the RNA movie in translation?

The ribosome moves down the mRNA in the 5’ to 3’ direction

Ribosomes have three different sites for the binding of tRNA:

Think ‘Arrive, Processing and Exit’ or A,P,E.

Which amino acid gets added to the polypeptide?

Each codon in the mRNA corresponds to one amino acid

If the anticodon on the tRNA is complementary to the codon on the mRNA:

The correct amino acid is at the ribosome and gets added

If the anticodon on the tRNA is not complementary to the codon on the mRNA

The correct amino acid is not at the ribosome; must wait for another tRNA with a complementary anticodon to arrive

Termination

• Elongation continues until the ribosome reaches a stop codon in the mRNA

• A protein called a release factor that causes the polypeptide chain to separate from the ribosome

Examples of stop codons

• UAG, UAA, or UGA

What happens after translation?

• The polypeptide folds up based on the arrangement of its amino acids (secondary and tertiary protein structure)

• Some polypeptides combine with others to make larger proteins (quaternary structure)

Where do the proteins go?

May be packaged at ER or modified and packaged at the Golgi

Protein Synthesis in Prokaryotes

• Transcription occurs in the cytoplasm

• No mRNA editing

• Transcription and translation occur simultaneously

Protein Synthesis in Eukaryotes

• Transcription occurs in the nucleus

• mRNA is edited prior translation

• Translation occurs after transcription is completed

Mutation

A change in the DNA sequence

Mutations can be caused by:

• Mutagens - external factors such as radiation and reactive chemicals (teratogens in a fetus)

• Errors in DNA replication (random)

• Errors in mitosis or meiosis (we talked about changes in chromosome number and structure in our meiosis notes)

Point mutations

• Also called a substitution

• A base is changed but the number of bases stays the same

Types of point mutations

• Nonsense - now code for a stop codon

• Missense - now codes for a different amino acid

• Silent - still codes for the same amino acid

How do mutations affect proteins?

Proteins fold up based on the properties of their amino acids; if a mutation results in an amino acid with a different property, the protein will have a different shape & function

Frameshift Mutations

The insertion or deletion of a base shifts the reading frame, changing all the codons after mutations

What type of mutation is this?

This is an insertion but not a frameshift because the codons were not regrouped as a result.

Are all mutations bad?

No, mutations are random and can be positive, negative, or neutral

Examples of positive, negative, and neutral mutations

• Positive - lactose tolerance in humans; antibiotic resistance in bacteria

• Negative - cystic fibrosis

• Neutral - eye color

How can some mutations be neutral?

• Could be silent

• Could be in an intron → not used to make proteins

Regulation of Gene Expression - Eukaryotes

• Even with the same DNA, not all cells in a multicellular organism look the same or do the same things (ex. Skin cells vs. stomach cells)

• Cells turn on the parts of DNA they need and off those they don’t → cell differentiation

Methods to regulate gene expression

• Regulate chromatin structure

• Regulate transcription initiation

• Post-transcriptional regulation

Regulating Chromatin Structure

DNA is wrapped around proteins called histones

Histone acetylation

Acetyl groups are added to histones, which prevents them from binding the DNA as tightly, making room for proteins to bind for transcription

DNA methylation

Methyl groups can attach to DNA bases, preventing transcription

Exons and Introns

Introns can be considered as intervening sequences, and exons as expressed sequences. There are an average of 8.8 exons and 7.8 introns per human gene.

Why are introns important?

They allow for alternative splicing, which in turn allows one gene to code for multiple transcripts and therefore serve multiple complex cellular functions.

What can introns affect?

Gene expression, the rate at which genes get turned on to make proteins and other non-coding RNA

Regulating Transcription Initiation

• Activators help RNA polymerase bind to the DNA, increasing transcription

• Repressors prevent RNA polymerase from binding to the DNA, preventing transcription

Transcription factors

Proteins that bind upstream of the gene

Post-transcriptional Regulation

RNA splicing

mRNA degradation

Initiation of translation

Protein processing and degradation

MicroRNAs

RNA Splicing

Different mRNA molecules are produced from the same primary transcript depending on which exons are used - one gene can code for more than one protein

mRNA degradation

Nuclease enzymes break down mRNA; lifespan of mRNA varies

Initiation of translation

If the ribosome does not form or mRNA cannot attach, translation does not occur

Protein processing and degradation

Many proteins need to be modified to become active usually via phosphorylation. They may be also be modified by the addition of ubiquitin, which triggers proteasomes to break down the protein

MicroRNAs

Can bind to mRNA, causing it to degrade or blocking it from being translated

Operon

A group of genes of related function