Protein Synthesis

1. 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: RNADNA sugar ribose deoxyribose bases A,U,C,G A,T,C,G strands single double

2. 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

3. 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.

4. 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

5. 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)

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

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

8. 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

10. 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

12. 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.

13. 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.

14. 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.

15. 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

16. 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)

17. 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

19. Transcription and Translation in Eukaryotes • prokaryotic mRNAs are used immediately after transcription • prokaryotes can transcribe and translate the same gene simultaneously.

20. 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.

21. 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

22. 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

23. 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)