Lecture 4 - Gene Expression

Central Dogma and transcription, Eukaryotic mRNA modifications, Translation, Mutations and translation

RNA polymerase

RNA polymerase

Enzyme similar to DNA polymerase that separates the strands of DNA to build a new RNA molecule 5' -> 3' that is complementary to one of the DNA strands

How is RNA polymerase different from DNA polymerase?

RNA polymerase doesn't need a helicase and can start a strand on its own

Promoter DNA Sequences

- mark the beginning of genes
- indicates the starting point and which way genes will go

TATA box Binding Protein (TBP)

eukaryotic transcription factor which binds to the TATA box (promoter) and recruits RNA polymerase

Rho-independent

- intrinsic transcription termination
- caused by hairpin in RNA transcript

Rho-dependent

- uses Rho translocase to remove RNA polymerase and DNA from transcript
- works similar to helicase - kicking off the polymerase and stopping replication

Eukaryotic cells modify RNA after transcription. What are those modifications?

end modifications:
- 5' methyl-G cap, 3' poly-A tail
- facilitate export from nucleus, protect from degradation, facilitate translation (ribosome recruitment)

Exons

- protein coding sequences
- expressed
- exit the nucleus

Introns

- non-protein coding
- intervening, "in the way"

RNA splicing

removes introns and connects exons before the coding sequence exits the nucleus

Are eukaryotic genes continuous or non-continuous?

non-continuous

Spliceosome

protein, RNA complex that carries out RNA splicing (as the name suggests)

snRNPs ("snurps")

- small nuclear ribonucleoproteins
- snRNA (small nuclear RNA) targets complex based on intron sequences

Eukaryotic mRNAs contain __ and __ segments

coding, non-coding

What do mRNA modifications help with?

- modifications allow mRNA to leave the nucleus and enter the cytoplasm
- the 5' cap and 3' tail aid in letting mRNA pass through the nuclear envelope as the envelope is very selective and will only allow specific things to pass
- once in cytoplasm, mRNA will swap out the cap and tails for new caps and tails that are associated with translation

Which way is mRNA read?

translation goes
5' -> 3' direction to build new proteins in the N-terminus to C-terminus direction

think
5 to 3, N to C

What groups and elements are amino acids made of?

amino group, carboxyl group,
carbon, hydrogen,
and an R group (side chain)

What attaches the the N-terminus?

an open amino group

What attaches to the C-terminus?

an open carboxyl group

Codon

set of three RNA nucleotides -> corresponds to just one amino acid

Characteristics of genetic code

- redundant: there's multiple codons per AA
- unambiguous: only one AA per codon
- evolutionary conserved

transfer RNA (tRNA)

- half nucleic acid, half amino acid
- structure is RNA folded on itself
- tRNA covalently attached to amino acids
- each tRNA has an anticodon
- 61 tRNAs in the cell (one for each codon minus the stop codons)

anticodon

- found on each tRNA
- base pairs with one out of 64 codons found in mRNA
- other end of the tRNA attaches to the correct amino acid of the codon

Amino-acyl tRNA synthase

- specifically recognizes amino acids and tRNA to make sure that the correct tRNA goes with the correct AA
- need ATP to load a tRNA

Ribosomes

- site of translation
- rRNA-protein complex
- two subunits come together to initiate translation

Three tRNA binding sites

A site: tRNA-amino acid binds here
P site: tRNA polypeptide binds here
E site: empty RNA exits from here

initiation

translation beings at an AUG codon

True/False: all polypeptides begin with the same amino acid

True, all begin with AUG

RIbosome recognition in bacteria

mRNA ribosome binding site (Shine-Delgarno Sequence) base pairs with rRNA from small subunit, lining up correct AUG start

Ribosome recognition in eukaryotes

- small ribosomal subunit binds 5' cap of mRNA
- Kozak sequence helps ribosomes find AUG

Stop codons

recruits release factor ( a protein factors, not tRNA) that terminates translation, and the translation complex is disassembled

Polyribosomes (polysomes)

multiple ribosomes simultaneously translate the same mRNA

True/False, and why: Transcription and translation happen at the same time in prokaryotic genes?

True
Prokaryotic genes don't have introns and the cells don't have a nucleus, so transcription and translation happen at the same time

Open reading frames (ORFs)

- help predict gene locations, especially in prokaryotes
- have a start codon and no premature stop codons

Mutations within protein-coding sequences

three types:
substitution
deletion
insertion

- mutations can be good, bad, or neutral
- can cause diseases but also driving force behind evolution

Substitution mutation

- one nucleotide is replaced by another
- missense: wrong amino acid
- silent: substitution that results in the same amino acid (b/c multiple codons result in the same AA)
-nonsense: premature stop codon created, causing a portion of the protein to be missing

Deletion and Insertion mutations

- deletion: one or more nucleotides are removed from the gene
- insertion: one or more nucleotides are added to a gene

- "frameshift" mutation
- usually changing a protein more drastically than substitution
- cause different set of codons to be read after mutation occurs