Chapter 17 Warm-Up Explain the contribution that Beadle and Tatum made to understanding the role of DNA. Compare and contrast DNA to RNA. What is the difference between replication, transcription and translation?

Chapter 17 Warm-Up Describe the steps in transcription. Contrast transcription in prokaryotes vs. eukaryotes. How many nucleotides are in an mRNA molecule to code for a protein with 200 amino acids?

Chapter 17 Warm-Up How does mRNA differ from pre-mRNA? What is the difference between introns and exons? Describe how spliceosomes modify mRNA.

Chapter 17 Warm-Up Describe the steps of translation. If the DNA sequence is: 3’ T A C G A T C A G 5’ the cDNA would be: the mRNA is: the tRNA is: the amino acid sequence is: How does the cell determine the ultimate destination of a polypeptide being synthesized?

Chapter 17 Warm-Up What is a frameshift mutation? How can they impact protein synthesis? Contrast a missense vs. nonsense mutation.

Chapter 17 Warm-Up Refer to page 327. Fill in the chart comparing prokaryotic and eukaryotic gene expression: Prokaryotes Eukaryotes

From Gene to Protein Chapter 17

What you need to know: The key terms: gene expression, transcription, and translation. The major events of transcription. How eukaryotic cells modify RNA after transcription. The steps to translation. How point mutations can change the amino acid sequence of a protein.

Concept 17.1: Genes specify proteins via transcription and translation How was the fundamental relationship between genes and proteins discovered?

Gene Expression: process by which DNA directs the synthesis of proteins (or RNAs) Old idea: one gene-one enzyme hypothesis Proposed by Beadle & Tatum – mutant mold experiments Function of a gene = dictate production of specific enzyme Newer idea: one gene-one polypeptide hypothesis Most accurate: one gene-one RNA molecule (which can be translated into a polypeptide)

one gene = one RNA molecule DNA RNA Nucleic acid composed of nucleotides Single-stranded Ribose=sugar Uracil Many different roles! Nucleic acid composed of nucleotides Double-stranded Deoxyribose=sugar Thymine Template for individual

RNA plays many roles in the cell pre-mRNA=precursor to mRNA, newly transcribed and not edited Messenger RNA (mRNA)= the edited version; carries the code from DNA that specifies amino acids Transfer RNA (tRNA)= carries a specific amino acid to ribosome based on its anticodon to mRNA codon Ribosomal RNA (rRNA)= makes up 60% of the ribosome; site of protein synthesis snRNA=small nuclear RNA; part of a spliceosome. Has structural and catalytic roles srpRNA=a signal recognition particle that binds to signal peptides RNAi= interference RNA; a regulatory molecule ribozyme= RNA molecule that functions as an enzyme

Flow of genetic information Central Dogma: DNA  RNA  protein Transcription: DNA  RNA Produces messenger RNA (mRNA) Occurs in the nucleus Translation: RNA  protein Ribosome are the site of translation

Flow of Genetic Information in Prokaryotes In prokaryotes, mRNA produced by transcription is immediately translated without more processing Why do you think that is? (Hint: what is missing?)

Flow of Genetic Information in Eukaryotes In a eukaryotic cell, the nuclear envelope separates transcription from translation Eukaryotic RNA transcripts are modified through RNA processing to yield finished mRNA

The Genetic Code How are the instructions for assembling amino acids into proteins encoded into DNA? There are 20 amino acids, but there are only four nucleotide bases in DNA So how many bases correspond to an amino acid?

Codons: Triplets of Bases The Genetic Code Codons: Triplets of Bases The flow of information from gene to protein is based on a triplet code: a series of non-overlapping, three-nucleotide words These triplets are the smallest units of uniform length that can code for all the amino acids Found on the mRNA strand Example: AGT at a particular position on a DNA strand results in the placement of the amino acid serine at the corresponding position of the polypeptide to be produced

The Genetic Code During transcription, a DNA strand called the template strand provides a template for ordering the sequence of nucleotides in an RNA transcript During translation, the mRNA base triplets, called codons, are read in the 5 to 3 direction Each codon specifies the amino acid to be placed at the corresponding position along a polypeptide

The Genetic Code 64 different codon combinations Redundancy: 1+ codons code for each of 20 AAs Reading frame: groups of 3 must be read in correct groupings This code is universal: all life forms use the same code.

Concept Check In RNA, _____ codon(s) translate to ______ amino acid(s) a. 1, 1 b. 3, 1 c. 3, 3 d. 1, 20 The sugar in RNA is _____, the sugar in DNA is _______ a. deoxyribose, ribose b. ribose, deoxyribose c. ribose, phosphate d. ribose, uracil Which of the following is found on RNA but not DNA? a. uracil b. deoxyribose c. phosphate d. adenine A stretch of chromosome that codes for a trait can be called a(n): a. chromatid b. replication fork c. gene d. base-pair How many different amino acids are there? A. 3 B. 20 C. 100 D. an infinite number

Concept 17.2: Transcription is the DNA-directed synthesis of RNA

Transcription Transcription unit: stretch of DNA that codes for a polypeptide or RNA (eg. tRNA, rRNA) RNA polymerase: Separates DNA strands and transcribes mRNA mRNA elongates in 5’  3’ direction Uracil (U) replaces thymine (T) when pairing to adenine (A) Attaches to promoter (start of gene) and stops at terminator (end of gene)

LE 17-7 Elongation Non-template strand of DNA RNA nucleotides RNA polymerase 3¢ 3¢ end 5¢ 5¢ Direction of transcription (“downstream”) Template strand of DNA Newly made RNA

Synthesis of an RNA Transcript The three stages of transcription: Initiation Elongation Termination

1. Initiation Bacteria: RNA polymerase binds directly to promoter in DNA

1. Initiation Eukaryotes: TATA box = DNA sequence (TATAAAA) upstream from promoter Transcription factors must recognize TATA box before RNA polymerase can bind to DNA promoter

2. Elongation RNA polymerase adds RNA nucleotides to the 3’ end of the growing chain (A-U, G-C)

2. Elongation As RNA polymerase moves, it untwists DNA, then rewinds it after mRNA is made As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time Transcription progresses at a rate of 60 nucleotides per second in eukaryotes A gene can be transcribed simultaneously by several RNA polymerases

3. Termination RNA polymerase transcribes a terminator sequence in DNA, then mRNA and polymerase detach. It is now called pre-mRNA for eukaryotes. Prokaryotes = mRNA ready for use

Flow of Genetic Information in Prokaryotes vs. Eukaryotes

Concept 17.3: Eukaryotic cells modify RNA after transcription Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm

Additions to pre-mRNA: 5’ cap (modified guanine) and 3’ poly-A tail (50- 520 A’s) are added Help export from nucleus, protect from enzyme degradation, assist ribosomes attach to the 5’ end

RNA Splicing Pre-mRNA has introns (noncoding sequences) and exons (codes for amino acids) Introns- stay IN the nucleus Exons- are EXpressed Splicing = introns cut out, exons joined together

RNA Splicing small nuclear ribonucleoproteins = snRNPs snRNP = snRNA + protein Pronounced “snurps” Recognize splice sites snRNPs join with other proteins to form a spliceosome Spliceosomes catalyze the process of removing introns and joining exons Ribozyme = RNA that acts as enzyme and can splice RNA

Why have introns? Some genes can encode more than one kind of polypeptide, depending on which segments are treated as exons during RNA splicing Alternative RNA Splicing: produce different combinations of exons One gene can make more than one polypeptide! 20,000 genes  100,000 polypeptides

Concept 17.4: Translation is the RNA-directed synthesis of a polypeptide

Components of Translation A cell translates an mRNA message into protein with the help of transfer RNA (tRNA) mRNA = message tRNA = interpreter Ribosome = site of translation

tRNA Transcribed in nucleus Specific to each amino acid Transfer AA to ribosomes Anticodon: pairs with complementary mRNA codon Base-pairing rules between 3rd base of codon & anticodon are not as strict. This is called wobble.

tRNA Accurate translation requires two steps: First step: a correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA synthetase Second step: a correct match between the tRNA anticodon and an mRNA codon Aminoacyl-tRNA-synthetase: enzyme that binds tRNA to specific amino acid

Ribosomes Ribosome = rRNA + proteins made in nucleolus 2 subunits Large and small

Ribosomes Active sites: A site: holds AA to be added P site: holds growing polypeptide chain E site: exit site for tRNA

Building a Polypeptide The three stages of translation: Initiation Elongation Termination All three stages require protein “factors” that aid in the translation process

1. Initiation- brings together mRNA, a tRNA with the first amino acid, and the two ribosomal subunits Small subunit binds to start codon (AUG) on mRNA tRNA carrying Met attaches to P site Proteins called initiation factors bring in the large subunit so the initiator tRNA occupies the P site

2. Elongation- amino acids are added one by one to the preceding amino acid

2. Elongation Codon recognition: tRNA anticodon matches codon in A site

2. Elongation Peptide bond formation: AA in A site forms bond with peptide in P site

2. Elongation Translocation: tRNA in A site moves to P site; tRNA in P site moves to E site (then exits)

2. Elongation Repeat over and over Codon recognition: tRNA anticodon matches codon in A site Translocation: tRNA in A site moves to P site; tRNA in P site moves to E site (then exits) Peptide bond formation: AA in A site forms bond with peptide in P site

3.Termination- when a stop codon in the mRNA reaches the A site of the ribosome Stop codon reached and translation stops Release factor binds to stop codon Causes the addition of a water instead of an amino acid polypeptide is released Ribosomal subunits dissociate

Polyribosomes A single mRNA can be translated by several ribosomes at the same time

Completing and Targeting the Functional Protein 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

Protein Folding During synthesis, polypeptide chain coils and folds spontaneously Chaperonin: protein that helps polypeptide fold correctly

Targeting Polypeptides to Specific Locations Types of Ribosomes-(are identical and can switch locations) Free ribosomes: synthesize proteins that stay in cytosol and function there Bound ribosomes (attached to ER): make proteins of endomembrane system (nuclear envelope, ER, Golgi, lysosomes, vacuoles, plasma membrane) & proteins for secretion Uses signal peptide to target location

Cellular “Zip Codes” Signal peptide: 20 AA at leading end of polypeptide determines destination Signal-recognition particle (SRP): binds to signal peptide, brings ribosome to ER

Concept Check- Quiz 2 RNA is synthesized on a DNA template in a process called ______, which utilizes the enzyme _______ a. translation, RNA polymerase b. transcription, DNA polymerase c. transcription, RNA polymerase d. replication, DNA polymerase Given the following DNA strand, which of the following is its complementary mRNA? G G A C T G A T T a. C C T G A C T A A b. C C U G A C U A A c. G G A C T G A T T d. T T A G T C A G G Once transcription has been completed, which of the following is NOT necessary for protein synethesis to occur? a. tRNA b. ribosomes c. mRNA d. DNA Which of the following is NOT a necessary component of translation? a. anticodon b. mRNA c. ligase d. amino acid Amino acids are joined together into a protein chain by which of the following? A. transfer RNA b. DNA polymerase c. hydrogen bonds d. messenger RNA

Concept 17.5: Point mutations can affect protein structure and function

The Central Dogma Mutations happen here Effects play out here

Mutations = changes in the genetic material of a cell Large scale mutations: chromosomal; always cause disorders or death nondisjunction, translocation, inversions, duplications, large deletions Point mutations: alter 1 base pair of a gene Base-pair substitutions – replace 1 with another Missense: different amino acid, more common Nonsense: stop codon, not amino acid Frameshift – mRNA read incorrectly; nonfunctional proteins Caused by insertions or deletions have a disastrous effect on the resulting protein more often than substitutions do

Substitution = Missense- different amino acid

Substitution = Nonsense- change an amino acid codon into a stop codon

Substitution = Silent (no effect)

Insertion = Frameshift Mutation

Deletion = Extensive missense, premature termination

Pulmonary hypertension Sickle Cell Disease Symptoms Anemia Pain Frequent infections Delayed growth Stroke Pulmonary hypertension Organ damage Blindness Jaundice gallstones Caused by a genetic defect Carried by 5% of humans Carried by up to 25% in some regions of Africa Life expectancy 42 in males 48 in females

Sickle-Cell Disease = Point Mutation

Mutation occurs in the beta chain – have them look at their amino acid structures and think about why the change may be important

Sickle cell hemoglobin forms long, inflexible chains

Comparison: Prokaryotes vs. Eukaryotes

Prokaryote vs. Eukaryote

Prokaryotes vs. Eukaryotes Transcription and translation both in cytoplasm DNA/RNA in cytoplasm RNA poly binds directly to promoter Transcription makes mRNA (not processed) No introns Transcription in nucleus; translation in cytoplasm DNA in nucleus, RNA travels in/out nucleus RNA poly binds to TATA box & transcription factors Transcription makes pre- mRNA  RNA processing  final mRNA Exons, introns (cut out)

A Summary of Protein Synthesis (p. 348) Most current definition for a gene: A region of DNA whose final product is either a polypeptide or an RNA molecule