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

transcription – a molecule of mRNA is made using DNA as a template 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 During transcription, one DNA strand, (template strand), provides a template for making an RNA molecule. 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.

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 (nucleoside 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

Transcription Video Clip

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.

Each ribosome has a large and a small subunit 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

Amino end of polypeptide Codon recognition 1 E E P A 3 mRNA 5 GTP Figure 14.19-1 Amino end of polypeptide 1 Codon recognition E 3 mRNA P site A site 5 GTP GDP  P i E P A Figure 14.19-1 The elongation cycle of translation (step 1) 16

Amino end of polypeptide Codon recognition Peptide bond formation 1 E Figure 14.19-2 Amino end of polypeptide 1 Codon recognition E 3 mRNA P site A site 5 GTP GDP  P i E P A Figure 14.19-2 The elongation cycle of translation (step 2) 2 Peptide bond formation E P A 17

Amino end of polypeptide Codon recognition Ribosome ready for Figure 14.19-3 Amino end of polypeptide 1 Codon recognition E 3 mRNA Ribosome ready for next aminoacyl tRNA P site A site 5 GTP GDP  P i E E P A P A Figure 14.19-3 The elongation cycle of translation (step 3) GDP  P i 2 Peptide bond formation 3 Translocation GTP E P A 18

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 Translation Video Clip

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)

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) RNA processing animation Protein Synthesis Video