Genetics - Gene Expression

Central Dogma

DNA → Transcription → mRNA → Translation → Polypeptide

RNA Polymerase

separates strands of DNA molecule to build new RNA molecule (5’ → 3’) that is complementary to one of the DNA strands

How is RNA polymerase different from DNA polymerase?

RNA Polymerase can:

start a strand from scratch, separate DNA strands as they go (doesn’t require helicase)

Promoter

DNA Sequence that marks the beginning of genes; attracts RNA polymerase to begin transcription there; defines direction to transcribe and which strand is the template

Consensus sequence

different regions of sequence that carry out the same task

Transcription factors

Proteins that assist and regulate transcription; bind to promoter and recruit RNA polymerase

TBP (TATA Box Binding Protein)

Binding protein; marks upstream start of transcription (TATA box located before start)

What does transcription termination rely on?

Transcription of DNA sequences that mark the ends of genes; once these sequences are transcribed, termination begins

What do eukaryotic cells do after transcription?

RNA modifications

RNA modifications at end of transcription

5’ Methyl-G cap

3’ poly-A tail

Purpose of end modifications

(5’ Methyl-G cap and 3’ poly-A tail)

Facilitate RNA export from nucleus, protects from degradation, facilitate translation via ribosome recruitment

Different segments in Eukaryotic mRNAs

Coding and non-coding sequences

Exons

Protein coding; expressed; exit the nucleus

Introns

Non-protein coding; intervening

Are eukaryotic genes continuous or non-continuous?

Non-continuous; introns and exons

RNA splicing

removing introns; connecting exons prior to exiting nucleus

Steps of RNA splicing

1. 5’ end of intron is cut → attached to branch point → forms lariat (lasso)

2. 3’ end of intron is cut → releases lariat, joining 2 exons

3. mature mRNA formed, ready to leave nucleus

Spliceosome

temporary protein-RNA complex that carries out RNA splicing

snRNPs (snurps)

small nuclear ribonucleoproteins

snRNA

small nuclear RNA

Targets complex based on intron sequences

What does RNA polymerase bring with it?

Modifying enzymes (ex. those that make poly-A tail and methyl-G cap)

Polyadenylation factors

proteins that create poly-A tail

Splicing factors

regulate RNA splicing; leave when it reaches introns

Capping factors

Create 5’ Methyl-G cap

Translation

use of mRNA to make polypeptides (chain of amino acids)

Messenger RNA (mRNA)

RNA transcripts that code for protein sequences

mRNA is read in the 5’ → 3’ direction to build new proteins in the ____ direction

N-Terminus → C-Terminus

What is at the N-terminus group and what is at the C-terminus group?

Amino group at N-terminus

Carboxyl group at C-terminus

Codon

Set of 3 RNA nucleotides that corresponds to one amino acid; each codon is read to make an amino acid

How are codons read?

One after the other; do not overlap with each other

How many codons are there in standard genetic code?

64

How many (essential) amino acids are there?

20

Characteristics of genetic code

Redundancy, but not ambiguity; Evolutionary conserved (same basic genetic code)

Redundant (in regards to genetic code)

multiple codons can code for the same amino acid

Unambiguous

only one amino acid per codon

Transfer RNA (tRNA)

Half nucleic acid, half amino acid

Functional, non-coding RNA

Anticodon

3 nucleotides on one end of tRNA to base pair with mRNA codon

What happens on the other end of tRNA (not the anticodon end)?

Binds to correct amino acid for mRNA codon

Amino-acyl tRNA synthetase

Recognizes amino acid and tRNA;

Attaches appropriate amino acid to tRNA

Ribosome

site of translation;

rRNA-protein complex → 2 subunits come together to initiate translation

3 tRNA binding sites on Ribosome

A site: tRNA-[amino acid]

P site: tRNA-[polypeptide]

E site: empty tRNA exits

Start codon

AUG

All polypeptides begin with the same amino acid:

AUG; Methionine (Met)

Initiation

Translation begins at an AUG codon

Shine-Delgarno Sequence (mRNA ribosome binding site) in bacteria

base pairs with rRNA from small-subunit; lines up correct AUG start codon at 5’ end

Kozak sequence (eukaryotes)

helps ribosome find AUG; small ribosomal subunit binds 5’ cap of mRNA

How many reading frames does each mRNA have?

3

Continued steps of translation

1. New tRNA with correct anticodon binds to next mRNA codon in A site

2. Growing amino acid chain connects to amino acid from new tRNA

3. Old tRNA leaves, message slides over to put tRNA holding peptide in P site → makes room for next tRNA in A site

4. Ribosome now ready for next aminoacyl tRNA

Stop codons

UAA, UAG, UGA

What do stop codons do?

Recruit protein factors that terminate translation; no tRNA binds to them

Termination

stop codon recruits release factor (protein, not tRNA) → translation complex disassembles

Polyribosomes (polysomes)

multiple ribosomes simultaneously translate the same mRNA;

one mRNA will have multiple ribosomes on it at once, all translating

Prokaryotic genes don’t have ___, and therefore don’t need to be spliced

Introns

Since prokaryotic cells don’t have a nucleus, ___ and ___ of a gene can happen at the same time

Transcription and Translation

Open reading frames (ORFs)

Any section of DNA/RNA with a start codon, amino acid codons, and stop codon

Contain start codon and no premature stop codons (stop codons are not in the middle)

ORFs help predict ____ , especially in prokaryotes

Gene locations; prokaryotes don’t have introns so it’s easier to predict where genes are using ORFs

Why are ORFs less useful for gene predictions in eukaryotic genomes?

Eukaryotic genomes still have introns. Removing introns can change whole sequence, since introns can possibly contain start/stop codons. Removing introns can change entire reading frame

Mutations

Changes to DNA sequences

Types of DNA mutations

Substitution

Deletion

Insertion

Substitution

one nucleotide is replaced by another

Deletion

One/more nucleotides are removed from gene

Insertion

One/more nucleotides are added to a gene

Mutations can be good or bad, but most are ___. They can cause diseases, but are also the driving force behind ___. 

Neutral

Evolution

Types of substitution mutations

Missense

Silent

Nonsense

Missense

Wrong amino acid is substituted in gene sequence

What type of DNA mutation is responsible for Sickle Cell Disease?

Missense (substitution)

Glu is replaced with Val

Why won’t every substitution result in a different amino acid on the protein?

Most amino acids are coded for by more than one codon

Ex. UUU and UUC both code for Phenylalanine (Phe)

Silent

Substitution mutation, but codes for the same amino acid as original codon

Substitutions create bigger problems if they create ___

Stop codons

Nonsense

Substitution mutation where stop codon is created

Causes rest of sequence (after stop codon) to not be read

What type of mutations are deletions and insertions classified as?

Frameshift mutations

Usually change a protein more drastically than substitutions

Why do deletions/insertions typically change proteins more drastically than substitutions?

not adding/deleting in a factor of 3, so frameshift occurs.

results in different set of codons to be read after mutation (different reading frame)

changes entire amino acid sequence