Genes, Translation and epigenetics (lectures 8+9)

What are the 3 steps in Transcription?

1. Initiation

2. Elongation

3. Termination

What is splicing?

Introns are removed

Extrinsic are joined together

Where does translation take place?

Cytoplasm of ribosomes

In simple terms, what does translation do?

Converts mRNA into protein

What are the components of translation?

• mRNA, tRNA, rRNA

• Ribosomes subunits (large and small)

• Codons and anticodons

What are the 3 stages of translation?

1. Initiation

2. Elongation

3. Termination

Same as transcription

Describe the initiation stage of translation

1. TRNA carries methionine to the SMALL ribosomal subunit

2. They bind to the 5’ end of the mRNA (5’ GTP cap)

3. They walk along the mRNA towards the 3’ direction to reach the start codon » AUG

What group is on the N-terminus?

Amino group

What group is on the C-terminus?

Carboxyl group

What are the 3 common stop codons?

UAG

UAA

UGA

What is the universal start codon?

AUG (Met)

When does protein folding occur?

During translation

What is epigenetics?

Heritable changes in gene expression

Does epigenetics change the DNA sequence?

No

Is epigenetics irreversible or reversible?

Reversible

What are 3 types of epigenetics?

DNA modifications

RNA modifications

Protein modifications

What are DNA modifications?

Chemical changes to DNA without altering its sequence

What is DNA methylation?

Additional of methyl groups to cytosine (CpG sites)

What is Hydroxymethylation?

Gene regulation and active DNA DEmethylation

What are the functions of DNA modifications?

• regulates gene expression (silencing)

• Development, X-chromosome inactivation, genomic imprinting

• Maintain genome stability

What are Histone Modifications?

Indirect DNA modifications through the CHROMATIN structure

What are post-transcriptional modifications?

Chemical changes to RNA molecules after transcription.

What are post-translational modifications (PTMs)?

Chemical modifications made to a protein after translation.

What are common types of PTMs?

• phosphorylation

• Glycosylation

• Ubiquitnation

• Acetylation/ Methylation

What are the types of post-transcriptional modifications? (5)

• 5’ capping

• 3’ polyadenylation

• RNA splicing

• RNA editing

• RNA methylation

What process creates mature mRNA?

RNA splicing

Describe the Termination process in Translation.

1. Stop codon enters A site

2. Stop codons are recognized by release factors

   1. Releases factors add an H2O molecule

   2. Cleaves polypeptide (C-terminus)

What does protein folding determine?

Function

What are the different types of DNA modifications?

• DNA methylation

• Hydroxymethylation

• DNA damage and repair mechanisms

• Histone modifications

What is 5’ capping?

Additional of Guanine to the 5’ end

What is 3’ Polyadenylation?

Addition of poly (A) tail to the 3’ end

What does RNA editing do?

Base changes that alter coding potential

What are the functions of post-transcriptional modifications?

• controls mRNA stability and lifespan

• Translation efficiency and localization

• Protein diversity through alternative splicing

• Responds to stress and developmental cues

What is the purpose of PTMs?

Regulates protein function, stability, localization, and interactions

Are PTMs reversible or irreversible?

Reversible

What proteins are phosphate groups typically added on during phosphorylation?

Serine (Ser)

Threonine (Thr)

Tyrosine (Tyr)

Kinases_____ phosphate groups, while phosphatases ____ phosphate groups.

ADD

REMOVE

What is Glycosylation?

Addition of carbohydrate chains to proteins

What are the 2 types of Glycosylation?

• N-linked» attaches to Asparagine (Asn)

• O-linked» attaches to Serine (Ser) or Threonine (Thr)

What is Ubiquitination?

Covalent attachment of ubiquities (small protein) to Lysine residues.

What are the 3 key enzymes in Ubiquitination?

1. E1» Ubiquitin-activating enzyme

2. E2» Ubiquitin-conjugating enzyme

3. E3» Ubiquitin ligase

What are the functions of Ubiquitination?

• Marks proteins for degradation

• Quality control

• DNA repair, immune signaling

Acetylation is the addition of an _____ group to a _____ residue, often on histones/regulatory proteins.

Acetyl

Lysine

What are the 2 enzymes involved in Acetylation? What do they do?

1. HATs (Histone Acetyltransferases)»»» ADDS acetyl groups

2. HDACs (Histone Deacetylases)»»» REMOVES acetyl groups

What are the functions Acetylation?

Alters chromatin structure+regulates gene expression

Involved in metabolism, transcription, DNA repair

Protein stability, localization, and interactions

Methylation is the addition of 1 or more ________ groups to _________, typically which 2 residues?

Methyl groups

Amino acids

Lysine and Arginine residues

What are the 2 enzymes involved in Methylation? What do they do?

1. Methyltransferases»»» ADD methyl groups

2. Demethylases»»» REMOVE methyl groups

What are non-coding RNAs (ncRNAs)?

RNA molecules that are NOT translated into proteins.

However, they regulate gene expression though epigenetic mechanisms.

What are the 4 types of ncRNAs?

• miRNAs (microRNAs)

• IncRNAs

• PiRNAs

• SiRNAs

What do miRNAs do?

Repress translation (silences)

Induce degradation

What do IncRNAs do?

Remodels chromatin

Transcriptional control

What do piRNAs do?

Silences transposons

What are the functions of Non-coding RNAs in epigenetics?

• recruit chromatin-modifying complexes

• DNA methylation and Histone modifications

• Silence/activate genes @transcriptional level

• Maintain genomic stability

What does miR-155 in Periodontal Disease?

Regulates inflammation by targeting genes in NF-kB pathway

Alters Histone acetylation and DNA methylation indirectly

Over-expressed in periodontitis »»» THERAPEUTIC target

What are a few key environmental influences on epigenetics?

Diet» nutrients impact DNA methylation.

Stress» alters Histone modifications and miRNA levels

Toxins» chemicals disrupt epigenetic patterns

Where in the cell is the genetic material?

Nucleus

What does the outer and inner membrane of the nuclear envelope consist of?

Outer » ribosomes

Inner » lamins (help structure of nucleus)

What goes in and out of the nuclear pores?

What goes in » nucleotides

What comes out » mRNA

What 2 structures does the nucleoplasm consist of?

• Nucleolus

• Chromatin

What is the function of the nucleolus?

Function » rRNA synthesis (ribosomes)

What is chromatin?

DNA wrapped around Histone proteins (resembles beads on a string)

What is euchromatin?

Less-condensed form of chromatin (LOOSE)

Under active transcription

What is heterochromatin?

HIGHLY condensed chromatin

NO transcription (inactive)

What is another term for “closed-chromatin”?

Hetero-chromatin

What is another term for “open chromatin”?

Euchromatin

What are the 2 components of a Nucleosome?

• DNA

• Histones

What is an octamer?

8 Histone proteins

2 H2A + 2 H2B + 2 H3 + 2 H4 proteins

DNA wraps around this complex

DNA wraps ______ times around a Histone octamer.

1.65

Which 2 proteins are histones rich in? What do they do?

Lysine (+) and Arginine (+)

They have POSITIVE charges which bind to the DNA, which is negatively charged.

What is the basic structural unit of chromatin?

Nucleosome (beads on a string)

What is the Histone H1 protein? What is its function?

Linker protein that connects Nucleosome via linker DNA.

MOST positively charged Histone

Helps compact DNA

What are DNA and RNA both composed of?

Nucleotides

What are the 3 structures of a nucleotide?

1. Nitrogenous base

2. Pentose sugar

3. Phosphate group

Which nitrogenous bases are purines?

Adenine (A)

Guanine (G)

Think AnGels are PURE

Which nitrogenous bases are pyrimidines?

Cytosine (C)

Thymine (T) » DNA

Uracil (U) » RNA

What is the difference of the pentose sugars between RNA and DNA?

RNA » Ribose

DNA » Deoxyribose

• MISSING Oxygen at the 2’ carbon

Which proteins for the core of a Nucleosome?

Histones

• H2A

• H2B

• H3

• H4

H1 is a LINKER protein » it is NOT in the core

What are the complementary base pairs in DNA?

A-T

G-C

What does the Nucleoside consist of?

Pentose sugar

Nitrogenous base

(NO PHOSPHATE GROUP)

In the sugar-phosphate backbone, nucleotides link together through which binding?

Phosphodiester bonds (2 ester bonds and a phosphate)

What is the directionality in the sugar-phosphate backbone?

5’ to 3’

Nitrogenous bases pair together through which binding?

Hydrogen binding

How many hydrogen bonds do A-T and G-C have?

A-T » 2 hydrogen bonds

G-C » 3 hydrogen bonds

• stronger binding

Is DNA parallel or anti-parallel?

Anti-parallel

The DNA double helix has how many nucleotides per turn?

10

Which 2 grooves does the DNA double helix have?

Major groove

Minor groove

What is the function of the major groove in the DNA double helix?

Binding site for enzymes/proteins

What does Dactinomycin do?

Binds to the MINOR groove

INHIBITS DNA replication

What is the function of the minor groove in the DNA double helix?

Binding site for drugs

Is DNA or RNA more stable?

DNA is more stable

RNA resides in both the nucleus and _____. Unlike, DNA, which only resides in the nucleus.

Cytoplasm

RNA » nucleus and cytoplasm

Which of the following RNA types is responsible for carrying amino acids to the ribosome during protein synthesis?

Type » tRNA

What is the gene involved in Dentinogenesis Imperfecta (DI)? How is the structure of this gene relevant?

DSPP

Mutations in coding region or regulatory sequences.

What are the genes involved in cleft lip and palate formation? How are the structure of these genes relevant?

MSX1

IRF6

PVRL1

TGFA

Variants in promotor/enhancer regions can alter craniofacial development.

What does DMP1 do in dental tissues? Where is it found?

Non-collagenous protein that plays a crucial role in tooth and bone mineralization.

Found in dentin, bone, and cementum.

What is Phosphorylation of DMP1 essential for ?

Calcium binding

Hydroxyapatite crystal formation

Regulation of Odontoblast differentiation

What can mutations to DMP1 lead to?

Dentinogenesis imperfecta-like defects

Hypophosphatemic rickets » enlarged pulp chambers, spontaneous abscesses

Negatively impacts tooth strength, structure, and susceptible to wear and infection.

How does Glycosylation play a critical role in oral microbial adhesion?

STRONG binding affinity to glycoproteins in acquired enamel pellicle.

Salivary glycoproteins (gp340/DMBT1) are heavily glycosylated » BINDING site for S.mutans

PROMOTES plaque formation by ENABLING bacterial adhesion.

Which salivary glycoproteins act protectively? How do they?

Mucins » MUC5B and MUC7

They TRAP microbes and promote clearance by swallowing.

INHIBITS microbial adhesion/prevents plaque

Which salivary glycoproteins are harmful? How are they?

Gp340/DMBT1

BINDING sites for bacteria like S.mutans » promotes plaque formation