From Gene to Protein

Gene Expression Process by which DNA directs the synthesis of a protein 2 stages transcription translation All organisms One 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 4 3 = – # 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-overlapping Compare the mRNA to the non-template strand How 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 transcription A. RNA polymerase binds to template strand of DNA  upstream downstream 

The promoter is upstream from the 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. tRNA 2 o structure is a cloverleaf CONCEPT CHECK How 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. Elongation Amino acids added to chain Peptide bonds between amino acids

3. Termination stop 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

3 rd position in mRNA codon “wobbles” Us and Cs can be matched with G in anticodon Rules of base pairing relaxed in the 3 rd position

After translation  ER  Golgi Post – translational modification