1. Exam Critical Concepts Chapter 17 & 20 Protein Synthesis and Biotech

2.  RNA is the intermediate between genes and the proteins for which they code  Protein synthesis is two steps 1. Transcription: synthesis of RNA from DNA ◦ produces messenger RNA (mRNA) 2. Translation: synthesis of a polypeptides ◦ occurs under the direction of mRNA  Ribosomes are the sites of translation Prokaryotes: mRNA is immediately translated without more processing Eukaryotes: nuclear envelope separates transcription from translation

3.  The central dogma: DNA to RNA to Protein  Codon: flow of information from gene to protein is based on this triplet code  20 amino acids make up all proteins  Each codon specifies the addition of one of 20 amino acids  Of the 64 triplets: 61 code for amino acids ◦ 3 triplets are “stop” signals to end translation  The genetic code is redundant but not ambiguous ◦ no codon specifies more than one amino acid  The genetic code is nearly universal, shared by the simplest bacteria to the most complex animals

4.  Template strand: one of the two DNA strands provides a template for ordering the sequence of nucleotides in RNA transcription  RNA polymerase: pries the DNA strands apart and hooks together the RNA nucleotides with the Same base pairing rules as DNA, except no thymine, uracil ◦ attaches at the promoter  Transcription unit: stretch of DNA that is transcribe  The three stages of transcription: 1.Initiation 2.Elongation 3.Termination

5.  Initiation: begins transcription  Transcription factors: mediate the binding of RNA polymerase and the initiation of transcription  Transcription initiation complex: the completed assembly of transcription factors and RNA polymerase II bound to a promoter  TATA box: promoter made up of thymine and adenine is where transcription begins  Elongation: RNA transcript is “built”  As RNA polymerase moves along the DNA, it untwists the double helix  A gene can be transcribed simultaneously by several RNA polymerases

6.  Termination: RNA transcript is completed  Different in bacteria and eukaryotes ◦ Bacteria: the polymerase stops transcription at the end of the terminator ◦ Eukaryotes: the polymerase continues transcription after the pre-mRNA is cleaved  Transcript processing: modify pre-mRNA before the genetic messages are dispatched to the cytoplasm ◦ both ends of the primary transcript are altered ◦ some interior parts of the molecule are cut out, and the other parts spliced together

7.  Each end of a pre-mRNA molecule is modified in a particular way: ◦ The 5 end receives a modified nucleotide 5 cap ◦ The 3 end gets a poly-A tail  These modifications share several functions: 1.They seem to facilitate the export of mRNA 2.They protect mRNA from hydrolytic enzymes 3.They help ribosomes attach to the 5 end  Introns: noncoding stretches of nucleotides that lie between coding regions  Exons: coding regions eventually expressed

8.  RNA splicing: removes introns and joins exons ◦ creating an mRNA molecule with a continuous coding sequence Pre-mRNA mRNA Coding segment Introns cut out and exons spliced together 5’ Cap Exon Intron 5’ 1 30 31104 ExonIntron 105 Exon 146 3’ Poly-A tail 5’ Cap 5’ UTR3’ UTR 1 146

9.  Spliceosomes: a variety of proteins and several small nuclear ribonucleoproteins (snRNPs) that recognize the splice sites  Ribozymes: catalytic RNA molecules that function as enzymes and can splice RNA  Alternative RNA splicing: some genes can encode more than one kind of polypeptide ◦ depending on which segments are treated as exons during RNA splicing ◦ the number of different proteins an organism can produce is much greater than its number of genes

10.  Importance of Introns: ◦ Domains: Proteins have modular architecture consisting of these discrete regions ◦ In many cases, different exons code for the different domains in a protein ◦ Exon shuffling: may result in the evolution of new proteins

11.  Translation: mRNA into proteins  Cell translates an mRNA message into protein with the help of transfer RNA (tRNA)  Molecules of tRNA are not identical: ◦ Each carries a specific amino acid on one end ◦ Anticodon on the other end ◦ base-pairs with a complementary codon on mRNA  Ribosomes: facilitate specific coupling of tRNA anticodons with mRNA codons in protein synthesis ◦ The two ribosomal subunits (large and small)

12. Polypeptide Ribosome Amino acids tRNA with amino acid attached tRNA Anticodon Trp Phe Gly Codons 3’ 5’ mRNA  Ribosome Binding sites: 1. P site: holds the tRNA that carries the growing polypeptide chain 2. A site: holds the tRNA that carries the next amino acid to be added to the chain 3. E site: exit site tRNAs leave the ribosome

13.  The three stages of translation: 1.Initiation 2.Elongation 3.Termination  Initiation: ◦ A small ribosomal subunit binds with mRNA and a special initiator tRNA ◦ The small subunit moves along the mRNA until it reaches the start codon (AUG) ◦ Initiation factors bring in the large subunit that completes the translation initiation complex

14.  Elongation: ◦ Amino acids are added one by one to the preceding amino acid ◦ Each addition involves proteins called elongation factors  Termination: ◦ Stop codon on mRNA reaches the ribosome A site ◦ A site accepts a protein called a release factor ◦ Release factor causes the addition of a water molecule instead of an amino acid ◦ This reaction releases the polypeptide, and the translation assembly then comes apart

15.  Polyribosome (or polysome): A number of ribosomes can translate a single mRNA simultaneously ◦ enable a cell to make many copies of a polypeptide very quickly  Often translation is not sufficient to make a functional protein  Polypeptide chains are modified after translation  Completed proteins are targeted to specific sites in the cell  During and after synthesis, a polypeptide chain spontaneously coils and folds into its three- dimensional shape

16.  Proteins may also require post-translational modifications before doing their job  Some polypeptides are activated by enzymes that cleave them  Other polypeptides come together to form the subunits of a protein  Two types of ribosomes: ◦ Free: in the cytosol ◦ Bound: attached to the ER

17.  Mutations: changes in the genetic material of a cell or virus ◦ The change of a single nucleotide in a DNA template strand can lead to the production of an abnormal protein  Point mutations: chemical changes in just one base pair of a gene 1.Base-pair substitutions 2.Base-pair insertions or deletions

18.  Insertions and deletions are additions or losses of nucleotide pairs in a gene ◦ have a disastrous effect on the resulting protein more often than substitutions do ◦ frameshift mutation: alter the reading frame  Mutagens: physical or chemical agents that can cause mutations  Examples: Formaldehyde, benzene, and chloloform

20. DNA Makes RNA Chapter 17 Protein Synthesis cont.

21. Ribosome large and small units mRNA tRNA Amino acids DNA makes RNA (transcription); RNA makes protein (translation) Chapter 17 Protein Synthesis cont.

22.  Recombinant DNA: nucleotide sequences from two different sources, combined in vitro  Genetic engineering: direct manipulation of genes for practical purposes  Biotechnology: manipulation of organisms or their genetic components to make useful products  Restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites ◦ Restriction fragments: cut pieces ◦ Sticky ends: cut DNA in a staggered way producing fragments that bond with complementary sticky ends of other fragments

23.  DNA ligase: enzyme that seals the bonds between restriction fragments  Polymerase chain reaction, PCR: can produce many copies of a specific target segment of DNA  Gel electrophoresis: uses a gel as a molecular sieve to separate nucleic acids or proteins by size ◦ DNA fragments produced by restriction enzymes ◦ A current is applied that causes charged molecules to move through the gel ◦ Molecules are sorted into “bands” by their size

24.  Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene  The procedure is also used to prepare pure samples of individual fragments  Gene therapy is the alteration of an afflicted individual’s genes ◦ holds great potential for treating disorders traceable to a single defective gene ◦ Vectors are used for delivery of genes into specific types of cells, for example bone marrow

25.  Transgenic animals: made by introducing genes from one species into the genome of another animal ◦ are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use ◦ “Pharm” plants are also being developed to make human proteins for medical use  Most public concern about possible hazards centers on genetically modified (GM) organisms used as food