From Gene to Protein Transcription and Translation

Black Urine Started it All Archibald Garrod hypothesized that genes code for enzymes Phenotypes are expressed via different chemical reactions in the cell Alkaptonuria is caused by the lack of an enzyme to break down alkapton

The Importance of Enzymes Eye color is determined by pigments- which are made by enzymes Most organic molecules are created and destroyed by chemical processes involving enzymes Led to one gene- one enzyme hypothesis

One Gene – One Polypeptide All enzymes are proteins Not all proteins are enzymes, and some gene products are not enzymes Each polypeptide subunit of a protein is created by one gene ONE GENE → ONE POLYPEPTIDE Requires 4 genes!

RNA (Ribonucleic Acid) DNA contains the code for the protein, but RNA does most of the work RNA contains ribose sugar, and A,C,G,U nucleotides Almost always single stranded Can fold and pair with itself (not always linear)

Transcription Overview RNA is “transcribed” from DNA DNA serves as a template to make a strand of messenger RNA (mRNA) that is complementary to the DNA strand

Translation Overview “Translation” of the mRNA blueprint into a functional polypeptide Takes place in ribosomes

These Processes All organisms ever studied use the same process of coding It evolved very early on, before our ancestors diverged We can use bacteria to synthesize human proteins Tobacco plant expressing firefly protein

Transcription

3 Stages Initiation Elongation Termination

Initiation RNA Polymerase binds to a region of the DNA called the promoter with the help of transcription factors Ensures we only transcribe the necessary gene The promoter contains what is called a TATA Box – an essential part of the promoter DNA sequence

Synthesis begins here

RNA Elongation RNA polymerase separates the DNA strands Synthesizes an mRNA strand complementary to the DNA Elongates in the 5' to 3' direction (begins at the 3' end of the DNA)

RNA Elongation RNA polymerase adds 60 nucleotides per second in eukaryotes DNA double helix reforms and the RNA peels away from the DNA molecule Multiple polymerases can transcribe at once

Termination A terminator signal in the DNA causes the end of transcription The polymerase breaks away and the mRNA is released

Eukaryotic Cells Modify RNA The product of transcription is called pre- RNA or primary transcript The pre-RNA is modified before leaving the nucleus and heading to translation Also called RNA processing

Alteration of mRNA ends The 5' end (beginning of RNA strand) has a “guanine cap” added to it  Protects mRNA and attaches to ribosomes The 3' end receives a poly A tail ( A nucleotides)  Facilitates leaving of nucleus, as well as protection for the end of the strand

RNA Splicing Long, noncoding sequences of RNA that are not necessary called introns Interspersed with exons, coding segments Introns are removed from the primary transcript, and exons are joined together

Splicing continued Accomplished by a complex of molecules called a spliceosome

What is the Purpose of Introns? Introns are not fully understood Different segments of the gene can be expressed at different times (some segments may be introns at times, and exons at other times) Some parts of the protein, or domains, function independently. Introns provide an opportunity for new combinations during crossing over

Nucleotide Triplets mRNA molecules can be viewed as a “sentence” of nucleotide triplets Called a codon Each codon will code for 1 amino acid

Codons form Reading Frames …UCAUGGCAGUCAGUC… Could be read as …UCA UGG CAG UCA GUC… Or …U CAU GGC AGU CAG UC… OR …UC AUG GCA GUC AGU C…

The Start Codon sets the reading frame …UCAUGGCAGUCAGUC… Translation starts at the start codon, to ensure the correct reading frame So the mRNA should be read as AUG GCA GUC AGU C…

Translation

Translation, The Synthesis of Polypeptides Messenger RNA carries code to ribosomes Transfer RNA or tRNA carries amino acids to the ribosomes Amino acids are assembled into a polypeptide

Amino Acids Proteins are made out of amino acids, connected by peptide bonds There are only 20 amino acids The amino acids consist of an amino group, a carboxyl group, an H atom and a side chain, which is what differs

Transfer RNA tRNA carries an amino acid on one end and an anticodon on the other The anticodon is complementary to codons on the mRNA i.e. anticodon UAC will bond to AUG (the start codon)

tRNAs carry the corresponding amino acid for each mRNA codon

Anticodon “wobbling” There are about 45 tRNAs The 3 rd nucleotide in the codon does not always have to match exactly Many codons that have the same first 2 nucleotides code for the same amino acid

Ribosomes Facilitates the production of proteins Consist of 2 subunits Subunits come together only when mRNA is present Smaller in prokaryotes

Ribosomal Binding Sites mRNA bonding site P site holds the growing polypeptide A site holds the next amino acid to be added E is the exit site for tRNA Ribosomes connect mRNA and tRNA

Translation Initiation Small ribosomal subunit binds to mRNA tRNA carrying Met binds to AUG (start codon) Large ribosomal subunit attaches

Elongation of Polypeptide Amino acids are added one by one The tRNA anticodon recognizes the mRNA codon, adding the correct amino acid The mRNA and tRNA are moved through the ribosome, so that the tRNA in the E site is released, the tRNA in P moves to E, and the tRNA in the A site moves to P (called translocation) The tRNA moving from P to E releases the polypeptide to the next tRNA

Termination Elongation continues until a stop codon reaches the A site A release factor binds to the stop codon Causes addition of water to polypeptide, separating polypeptide from tRNA Ribosome and mRNA then separate

Many Ribosomes Work at Once As mRNA leaves one ribosome, it can begin synthesizing proteins in another ribosome Many ribosomes can operate on one mRNA, forming polyribosome complexes

Recall Protein Structure Genes determine the primary structure (sequence of AAs) Chaperone proteins may help form particular structures Protein folds and coils Also can be modified by enzymes before becoming functional

The Genetic Code AUG codes for methionine, but also serves as the “start” signal All polypeptides begin with Met which may be removed later UAA, UAG and UGA serve as “stop” codons and do not code for amino acids mRNA Amino acid

All 64 Codons

A Few Notes Notice that there is redundancy in the code – multiple codons code for most amino acids, but there is no ambiguity! Each triplet codes for only one amino acid The correct reading frame is essential, that is reading the code in the correct order. This is why we always start with AUG A U G U A G U C C G U A C G T U There are 3 possible reading frames, only one of which is correct! We would get completely different proteins