Protein Synthesis• The genetic code – the sequence of nucleotides

in DNA – is ultimately translated into the sequence of amino acids in proteins – gene expression

• in general, one gene encodes information for one protein (can be structural or enzymatic) – one-gene, one-protein hypothesis

• DNA does not directly synthesize proteins• RNA acts as an intermediary between DNA and

protein – polymer of nucleotides but has several important differences:

RNA DNA

sugar ribose deoxyribose bases A,U,C,G A,T,C,G strands single double

Protein synthesis occurs in two major steps – transcription and translation

• transcription – a molecule of mRNA is made using DNA as a template

• translation – the molecule of mRNA is used to make the protein

Overview of Protein Synthesis

– Complementary RNA molecule is made using base-pairing rules, except uracil pairs with adenine.

• During translation, blocks of three nucleotides (codons are decoded into a sequence of amino acids.

•During transcription, one DNA strand, (template strand), provides a template for making an RNA molecule.

Three types of RNA

1. messenger RNA (mRNA) – the “copy” of the DNA that is used to specify the sequence of amino acids in the protein

• mRNA nucleotides are read in groups of three called codons

• each codon codes for a specific amino acid

2. transfer RNA (tRNA) – bring amino acids to the ribosome during protein synthesis

• each tRNA carries a specific type of amino acid

• each tRNA can recognize a specific mRNA codon because it has a complementary anticodon (sequence of three bases that associates with the codon by base pairing)

•Each amino acid is joined to the correct tRNA by aminoacyl-tRNA synthetase•Aminoacyl tRNA – tRNA with it’s amino acid attached

3. ribosomal RNA (rRNA) – forms part of the ribosome

Transcription • synthesis of RNA using DNA as a

template• most RNA is synthesized by DNA-

dependent RNA polymerases • enzymes that require DNA as a template• similar to DNA polymerases• synthesize RNA in a 5’ to 3’ direction• use nucleotides with three phosphate

groups as substrates (nucleotide triphosphates), removing two of the phosphates as the subunits are linked together (just like DNA synthesis)

• the transcibed strand of DNA and the complementary RNA strand are antiparallel

• Transcription begins with an RNA polymerase attaching to a DNA sequence called the promoter (promoter is not transcribed) – marks the beginning of the gene

• RNA polymerase unwinds the DNA strand• only one of the strands of DNA is

transcribed – called the transcribed strand, template strand, or antisense strand

• The strand that is NOT transcribed is the sense strand

• RNA polymerase continues down the gene synthesizing a single strand of mRNA through base-pairing (A matches with U) until it reaches a termination signal

Translation – protein synthesis • In the process of

translation, a cell interprets a series of codons along a mRNA molecule.

• Transfer RNA (tRNA) transfers amino acids from the cytoplasm’s pool to a ribosome.

• The ribosome adds each amino acid carried by tRNA to the growing end of the polypeptide chain.

Ribosome Structure• Each ribosome has a large and a small subunit• These are composed of proteins and ribosomal RNA

(rRNA)• Each ribosome has a binding site for mRNA and

three binding sites for tRNA molecules.– The P site holds the tRNA carrying the growing

polypeptide chain.– The A site carries the tRNA with the next amino acid.– Discharged tRNAs leave the ribosome at the E site.

• Translation occurs in steps called: initiation, elongation, and termination

• Step1. Initiation brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits.– First, a small ribosomal subunit binds with mRNA and a

special initiator tRNA, which carries methionine and attaches to the start codon.

– in all organisms, protein synthesis begins with the codon AUG (codes for methionine)

– Initiation factors bring in the large subunit which closes in a way that the initiator tRNA occupies the P site.

Step 2. Elongation – the addition of amino acids to the growing polypeptide chain

• initiator tRNA is bound to the P site of the ribosome

• A site is filled with the next tRNA -specified by the codon (tRNA anticodon matches with codon by base-pairing)

• the amino acids are linked together (peptide bond)

• the tRNA in the P site moves to E site to be released and the ribosome moves down freeing up the A site

• the ribosome moves in a 5’ to 3’ direction as the mRNA is translated

•The genetic code is series of codons; read one triplet at a time•genetic code is redundant – certain amino acids are specified by more than one codon – 64 possible codons but only 20 amino acids•61 codons specify amino acids – three do not (UAA, UGA, and UAG are all stop codons – code for nothing)

Step 3. Termination – ribosome reaches the termination codon (stop codon) at the end of the sequence – stop codon does not code for an amino acid

Transcription and Translation in Eukaryotes • prokaryotic

mRNAs are used immediately after transcription

• prokaryotes can transcribe and translate the same gene simultaneously.

• eukaroytic mRNAs must go through further processing – posttranscriptional modification and processing:

• At the 5’ end of the pre-mRNA molecule, a modified form of guanine is added, the 5’ cap.– This helps protect mRNA from hydrolytic enzymes.– It also functions as an “attach here” signal for

ribosomes.

• At the 3’ end, an enzyme adds 50 to 250 adenine nucleotides, the poly(A) tail.

• eukaryotic genes have interrupted coding sequences – they contain long sequences of bases within the protein-coding sequences that do not code for amino acids in the final protein

• noncoding regions within the genes are called introns (intervening sequences)

• protein-coding sequences are called exons (expressed sequences)

• a eukaryotic gene may have multiple introns and exons

• the entire gene that is transcribed as a large mRNA molecule is called a precusor mRNA or pre-mRNA – contains both introns and exons

• a functional mRNA may be 1/3 the length of the pre-mRNA

• In order for a pre-mRNA to become a function message, it must be capped, have a poly-A tail added, have the introns removed, and have the exons spliced together

• excision of introns and splicing of exons catalyzed by snRNPs (small nuclear ribonucleoprotein complexes)