From Gene to Protein

Gene Expression

• Process by which DNA directs the synthesis of a protein

• 2 stages transcription translation

• All organismsOne gene one protein

1. Transcription of DNA

• Gene– Composed of DNA– 1000s of base pairs long– Sequence of G, A, T, C is a code

A portion of the insulin gene

ACATACATGTAGCAACGAAACCTGAACATGCGAACATTGACACAACAACAAUGUAGCCA

• A gene is transcribed into mRNA– One DNA strand is a template– Follow base complementation using G, A, U, C

Which strand is the template strand?

• This mRNA is referred to as a “transcript”• Single stranded• Uracil nucleotide (not thymine)• ONLY gene of interest is transcribed• mRNA will leave the nucleus for translation

step

• mRNA (transcript) leaves nucleus • Translated to protein in cytoplasm

• What organelle translates mRNA to protein?

• How many amino acids?• How many amino acids long is a

protein?• How could 10 identical protein

molecules be translated from 1 mRNA?• What happens to mRNA when the cell

has finished with it?• Can the cell transcribe more than one

gene at a time?

2. Translation of mRNA• Nucleotides (G, A, U, C) translated to amino acids• Ribosomes assemble amino acids to form polypeptide• What is the difference between a polypeptide and a protein?• What is the length difference between a chromosome and an

mRNA transcript?• Why do researchers use frog embryos to study translation?

Differences between prokaryotes and eukaryotes

• Proks – No nucleus– Translation can begin while transcription is still in

progress

• Euks– Nuclear envelope– Transcript is modified before leaving nucleus• Primary transcript mRNA mRNA

– Translation in cytoplasm

Central Dogma

DNA mRNA protein

(F. Crick 1956)

The genetic code

Triplet code 43 = – # combinations of 3 using G, A, T, C– Can code for all 20 aa

• Each triplet codes for a certain amino acid

Codon table (1960’s Nirenberg and Khorana)

• 3 stop codons• 1 start codon• Redundant• Nearly universal

Which strand of DNA is the template?Identify the codons (triplets)Code is non-overlappingCompare the mRNA to the non-template strandHow many codons are needed for a 20 amino acid

long protein?How many nucleotides?

The reading frame

AUG UGG GUU GGC UCA Amino acids? met –trp- phe-gly-ser

Transcription in more detail

1. Initiation of transcriptionA. RNA polymerase binds to template strand of DNA

upstream ----------------------------------------------downstream

The promoter is upstream fromthe start site for transcription

B. Transcription factors (proteins) are required for RNA pol to bind

C. mRNA begins to be synthesized

(RNA pol does not need a primer)

2. Elongation of transcript

– RNA pol synthesizes 5’ 3’– More than one

transcript made

3. Termination RNA pol detaches

Translation summary

Translation in more detail

• Cytoplasm• The players: • Transfer RNA tRNA• Ribosomes• mRNA• Amino acids

1. tRNA2o structure is a cloverleaf

CONCEPT CHECKHow many bases is a tRNA?How many bases is an anticodon?Where is the amino acid site with respect to the anticodon?Why does the tRNA look to be double stranded?What does the “t” mean in tRNA?

• A charged tRNA carries an amino acid

• Contains an anticodon– Complementary to a

codon

Note the CCA

• rRNA + protein• Large and small subunits

2. Ribosome

3 binding sites on ribosome

• P site holds tRNA• A site for tRNA with next amino acid• E site allows tRNA to leave• Note: large and small • subunits

Ribosomes

adds each aa from tRNA to growing polypeptide chain

up to 100,000s per cell

Activity overview of protein synthesis

Stages of translation

1. Initiation• Ribosome scans

mRNA for AUG start

• tRNA brings met

2. ElongationAmino acids added to chainPeptide bonds between amino acids

3. Terminationstop codon

Polypeptide released

• Polyribosomes• One mRNA

translated by many ribosomes

Wobble hypothesis

• How many sense codons?• Stop codons?• BUT……………only 45 tRNAs• Some tRNAs recognize more than one codon

3rd position in mRNA codon “wobbles”Us and Cs can be matched with G in anticodon

Rules of base pairing relaxed in the 3rd position

After translation ER Golgi

Post – translational modification