10.1 One Gene Encodes One Polypeptide

• One Gene One Polypeptide: Each gene in the genome encodes only a single polypeptide (1:1 ratio of genes to polypeptides)

• Transcription: The synthesis of RNA using one strand of DNA as a template.

• Translation: The synthesis of a protein (polypeptide); Takes place on ribosomes, using the information encoded in messenger RNA.

• Central Dogma of Molecular Biology: The premise that information flows from DNA to RNA to polypeptide (protein).

• Some RNAs do not encode proteins.

• mRNA, rRna, and tRNA: Most abundant RNA types

• mRNA: Encodes protein information that comes from a template strand of DNA; leaves the nucleous.

  • Only about 5% of all RNA in a cell

• Coding Strand: One of the two strands of DNA that for a particular gene specifies the amino acids in a protein; Same base sequence as transcribed RNA but with Ts instead of Us.

• Template Strand: The DNA strand that is transcribed to create an RMA transcript; Also refers to a strand of RNA that is used to create a complementary RNA.

• mRNA needs to be processed before translation in eukaryotes but not prokaryotes (mRNA is immediately ready).

• rRNA: Several species of RNA that are incorporated into the ribosome; Involved in peptide bond formation.

  • Actually forms the polypeptide

  • Makes up about 80% of all RNA in a cell

• tRNA: A family of folded RNA molecules; each carries a specific amino acid and anticodon that will pair with the complementary codon in mRNA during translation; recognizes which amino acid needs to be added next

  • Make up 15% of all RNA

10.2 Gene Expression Begins with Transcription of DNA into RNA

• RNA polymerase read the DNA template in the 3 to 5 direction and synthesize the RNA strand in the 5 to 3 direction.

  • Do not need a primer to begin

  • RNA can technically start synthesizing anywhere, but DNA sequences and certain proteins tell RNA where to stand.

  • Only on RNA polymerase in bacteria and archea, but several in eukaryotes

• Steps of Transcription

  • Initiation

    • Requires a promoter (special region of DNA to which the RNA polymerase binds) to tell RNA polymerase where to begin and which of the 2 strands to transcribe

    • Sigma Factors: a protein that binds to RNA polymerase, allowing the complex to bind to and stimulate the transcription of a specific class of genes

    • Transcription Factors: Proteins that assemble on a eukaryotic chromosome, allowing RNA polymerase II to perform transcription.

  • Elongation

    • RNA polymerasse synthesizes the RNA strand

    • First nucleotide in the new RNA forms on the 5 end

    • RNA transcript is antiparallel to the DNA template

    • Transcription occurs when RNA polymerase catalyzes the formatio nof phosphodiester bonds between added nucleotides and the growing RNA chain, releasing pyrophosphate in the process

    • Mistakes in transcription are generally okay

      • RNAs aren’t very long

      • RNA errors aren’t passed on to a new generation of RNA because they’re constantly new

      • Many copies of RNA are synthesized from a gene,

  • Termination

    • Multiple proteins involved

• Coding Regions: The nucleotide sequences in a gene that directly specify amino acids in a protein.

• Prokaryotic vs Eukaryotic Gene Expression

  • Prokaryotes

    • Transcription/translation happen at the same time in cytosol

    • DNA sequence not usually not interrupted by introns

    • Usually no modification after initial transcription

  • Eukaryotes

    • Transcription in the nucleus, then translation in the cytosol

    • Transcribed regions often interrupted by noncoding introns

    • Introns spliced out of pre-mRNAl 5’ cap and 3’ poly A tail added to mRNA

• Introns: A portion of a gene within the coding region that is transcribed into pre-mRNA but is spliced out prior to translation

  • If not removed, a nonfunctional protein will be made

• Exons: A portion of a DNA molecule, in eukaryotes, that is present in the mature mRNA and codes for part of a polypeptide.

• Both introns and exons occur in the primary mRNA transcript

  • Pre-mRNA: The initial gene transcript before it is modified to produce functional mRNA; Also known as the primary transcript.

    • Process includes cutting introns out of pre-mRNA and splicing exons together

    • Introns interrupt, but do not scramble, the DNA sequence of a gene.

• The primary transcript of a eukaryotic gene is modified in several ways before it leaves the nucleus: introns are removed, and both ends of the pre-mRNA are chemically modified

• RNA Splicing: The last stage of RNA processing in eukaryotes, in which the transcripts of introns are excised through the action of small nuclear ribonucleoprotein particles (snRNP).

  • Requires consensus sequences

  • Consensus Sequences: Short stretches of DNA that appear, with little variation, in many different genes.

  • Branch point: A conserved sequence (A followed by several pyrimidines) in the interior of an intron that is used during intron splicing to attach the 5’ end of the intron.

  • snRNPs: A complex of an enzyme and a small nuclear RNA molecule, functioning in RNA splicing.

    • Spliceosome: An RNA–protein complex that splices out introns from eukaryotic pre-mRNAs

      • Removes intron in two steps, first cutting the 5’ end and joining it to the branch site, then the 3’ end is cut and the two exons are joined.

• More pre-mRNA processing

  • 5’ Cap: A chemically modified GTP added to the 5’ end of mRNA; facilitates binding of mRNA to ribosome and prevents mRNA breakdown.

    • Added to 5’

  • Poly A Tail: A long sequence of adenine nucleotides (50–250) added after transcription to the 3’ end of most eukaryotic cells.

    • Helps export mRNA from nucleus, bind proteins, and makes mRNA stable

10.3 The Rules for Translation of RNA into Amino Acids are contained in the Genetic Code

• Codons: Three nucleotides in messenger RNA that direct the placement of a particular amino acid into a polypeptide chain.

• Genetic Code: The set of instructions, in the form of nucleotide triplets, that translates a linear sequence of nucleotides in mRNA into a linear sequence of amino acids in a protein.

• Sense Codons: A sequence of three nucleotides in an mRNA that encodes a particular amino acid.

• Almost all amino acids are encoded by at least two codons.

• 64 possible codons, but only 61 encode amino acids

• Translation is terminated by a nonsense codon

• Start Codon: The mRNA triplet (AUG) that acts as a signal for the beginning of translation at the ribosome.

• Nonsense Codons: Any of the three mRNA codons that signal the end of protein translation at the ribosome: UAG, UGA, UAA; also called stop codon.

• When an amino acid is encoded by four codons, the first two letters are always the same.

• When more than one codon encodes a single amino acid, the codons are almost always a single substitution away from one another (AAA versus AAG for example).

  • This means some mutations that cause a change in a codon do not alter the encoded amino acid—called synonymous and do not alter phenotype

• Transgenic organisms: An organism engineered to contain, and usually express, a gene from another organism.

• Synonymous mutations: When a DNA substitution alters the codon but does not alter the encoded amino acid; occur because of the degeneracy of the genetic code.

• Missense mutations A change in a gene’s sequence that changes the amino acid at that site in the encoded protein; usually causes a single amino acid change in the protein, which may or may not cause a change in function.

• Nonsense mutations: A change from a sense codon to a stop (nonsense) codon, causing a premature termination of translation and a shortened protein; usually also loss of function.

• Loss-of-stop mutations: A change from a stop codon to a sense codon, causing additional amino acids to be added to the end of the protein; effects depend on how many amino acids are added to the end of the protein and how important that part of the protein is to function.

• Frame-shift mutations: The addition or deletion of a single or two adjacent nucleotides in a gene’s sequence; Results in the misreading of mRNA during translation and the production of a nonfunctional protein.

  • Usually loss-of-function mutations because they affect so many amino acids in the protein.

10.4 RNA is Translated into Amino Acids by Ribosomes

• Two things must happen so that a protein made is the one specified by the mRNA:

  • (1) a tRNA must chemically read each mRNA codon correctly

  • (2) the tRNA must deliver the amino acid that corresponds to the mRNA codon

• There is at least 1 specific tRNA molecule for each of the 20 amino acids.

• Each tRNA has three functions fulfilled by its structure and base sequence

  • tRNAs bind to particular amino acids

    • Covalent bond

    • 3’ end of tRNA

  • tRNAs bind to mRNA

    • Anticodon: The three nucleotides in transfer RNA that pair with a complementary triplet (a codon) in messenger RNA

    • Codon & anticodon bind by hydrogen bonds and run antiparallel

  • tRNAs interact with ribosomes

    • Noncovalent interactions

• If the codon and anticodon interacted with complementary base pairs, the cell would need 61 different tRNA molecules, each with a different translaanticodon

• There are actually fewer anticodon sequences than codon sequences

  • Possible because the base pairing at the 3rd position (3’ end codon and 5’ end anticodon) is not strictly complementary

  • Wobble - certain bases in the third position of the anticodon are able to pair with more than just their normal partner

  • A single tRNA can pair with two codons and start with the same two letters but end in A/G or C/T

  • tRNA synthetases: highly specific enzymes that only bind to one amino acid and one corresponding tRNA, binds using energy

• In eukaryotes, the large subunit of a ribosome consists of 3 different ribosomal RNA molecules and about 49 protein molecules arranged in a precise pattern.The small subunit has 1 rRNA and 33 proteins

• When not active in the translation of mRNA the ribosome exists as two separate subunits.

• On the ribosome’s large subunit, there are 3 sites tRNA can bind to

  • A (amino acid), P (polypeptide), and E (exit) sites

• Ribosome moves along mRNA in the 3’ direction

• To double check, any tRNA that does not form H bonds with all three codon bases is ejected

• Three steps of translation: initiation, elongation, termination

  • Initiation

    • Initiation Complex: In protein translation, a combination of a small ribosomal subunit, an mRNA molecule, and the tRNA charged with the first amino acid coded for by the mRNA; formed at the onset of translation.

    • Once the small subunit is in place, the anticodon of a methionine-charged tRNA binds to the start codon by complementary base pairing to complete the initiation complex.

    • the first amino acid in a new polypeptide chain is always methionine.

    • Initiation factors: The proteins involved in helping to assemble the translation initiation complex.

  • Elongation

    • Where synthesis of the peptide occurs

    • Large subunit then breaks the bond between the methionine and its tRNA in the P site and then catalyzes the formation of a peptide bod between the methionine and the amino acid attached to the tRNA in the A site

    • Ribozyme: An RNA molecule with catalyctic activity.

    • Polypeptides grow from amino to carboxyl

    • Continues until the robosome shifts and a stop codon enters the A site

10.5 Proteins are Sometimes Modified after Translation

• Protein synthesis always begins on free ribosomes floating in the cytosol, which is the “default” location for a protein

• Signal Sequence: The sequence within a protein that directs the protein to a particular organelle.

• For some proteins

  • As translation proceeds, the polypeptide enters the RER and an enzyme cleaves off the signal sequence.

  • Proteins in the RER that lack signal sequences for destinations within the endomembrane system are usually secreted from the cell via vesicles that fuse with the cell membrane

• For other proteins

  • Translation completed in the cytosol, and then a signal sequence is bound by other proteins that move it to the correct organelle

• Most protein modifications occur after translation

  • The removal of hte signal sequence inside the RER occurs during

  • Removal of the initiator methionine also happens during

• Post-translatio Modifications in Proteins

  • Proteolysis: Cutting a polypeptide chain; large polyproteins cannot function unless cut

  • Glycosylation: Addition of carbohydrates to proteins to form glycoproteins; helps direct some proteins to lysosomes, or help for conformation/regcognition functions at the cell surface

  • Phosphorylation: the additon of phosphate groups to proteins, catalyzed by protein kinases; helps with cell signaling