Chapter 12: Protein Synthesis and RNA: how genes are expressed

DNA’s genetic info has code for all proteins needed by a cell

All organisms share fundamental genetic similarities, share the SAME genetic code

Gene expression = complex series of events by which the information contained in the sequence of bases in DNA is decoded and used to specify the makeup of the proteins in the cell

Proteins affect the cell phenotype

Transcription = RNA molecules are synthesized as complementary copies of a DNA template ( pic p 266)

Translation = protein synthesis

RNA, ribonucleic acid = intermediary between DNA and protein

· polynucleotide (lots of nucleotides linked together)

· single stranded (usually)

· sugar = ribose, 5 carbon; deoxyribose has 1 less oxygen that ribose

· Uracil in RNA, thymine in DNA. Both pyrimidines, so RNA has A=T

Transcription: DNA is copied (transcribed) into RNA

· sequence of bases in RNA is determined by complementary base pairing with bases in DNA

· mRNA, messenger RNA takes DNA info and uses it to make protein

Translation: translate info from mRNA into protein amino acid sequence

· nucleotide language translated into amino acid language

Codon = sequence of 3 consecutive bases in mRNA that specifies one amino acid in a protein

· triplet code (3 nucleotides / amino acid)

· Table 12-1!

tRNA, transfer RNA = adapter between the nucleotide codon and the amino acid

· tRNA links with specific amino acid

· tRNA recognizes the mRNA codon for the exact amino acid

· tRNA has anticodon sequence of 3 nucleotides complementary to the mRNA 3 nucleotide codon

· To translate, the tRNA anticodon hydrogen bonds to the complementary base pair of mRNA

· The amino acids on the tRNA link in the order as per the mRNA codon order.

· Ribosomes (remember?) = organelles of rRNA and proteins, made of 2 subunits

· ribosome attaches to mRNA, moves down mRNA as tRNA’s bring in the appropriate amino acids

· Amino acids are thus linked in the order as per the mRNA order.

Most RNA made by DNA-dependent RNA polymerase enzymes in all cells

· DNA template

· synthesize nucleic acids 5’ to 3’

· substrate = nucleotide triphosphate with lots of energy (like ATP), 2 PO4 lost when bind n’tide

· For complementary base pairing to work, the 2 strands are antiparallel: transcribed strand of DNA is antiparallel to the complementary RNA strand

Promoter = DNA sequence to which RNA polymerase must bind to begin transcription

· RNA synthesis doesn’t need a primer (unlike DNA)

· first 5’ nucleotide is triphosphate; others added lose 2 PO4 groups when added to 3’ end

· sugar-phosphate backbone

· last nucleotide has exposed 3’ OH hydroxyl group

· termination has “stop” specific base sequence

Upstream = toward the 3’ end of the transcribed DNA strand

· toward the 5’ end of the mRNA sequence (see yellow box p 269)

· downstream = toward the 5’ end of the transcribed DNA strand, or toward the 3’ end of RNA

Start transcribing: RNA polymerase recognizes a specific promoter sequence of bases UPSTREAM from the protein-coding sequence

· E. coli bacteria promoter 40 bases long

· Once RNA pol recognizes promoter, RNA pol unwinds helix and starts

· Usually only ONE strand of DNA is transcribed in the protein-coding region of DNA

· transcribed strand aka template strand, which is complementary to the sequence in mRNA

· If DNA = ATC, then mRNA = UAG, and t RNA = AUC. because have to be complementary

· If nontemplate strand were transcribed, a different, nonfunctional protein made.

· At different areas along the DNA, one strand can be transcribed for one mRNA, while later in the sequence, that strand may be nontranscribed.

mRNA = extra base sequences (bacteria, fig 12-5)

· leader sequence at 3’ end, noncoding, but recognition signal for ribosome binding

· Coding sequence = messages for protein

· Termination / stop codon at end of coding sequence: UAA, UGA, UAG. Don’t code for amino acid

· Noncoding 3’ trailing sequence of various lengths

Translation: protein synthesis converts the 4 base nucleotides organized into triplet codons into the 20 amino acid alphabet of proteins.

· more than 100 kinds of macromolecules involved

· tRNA’s bring the appropriate amino acids to match the mRNA sequence

· the amino acids link in peptide bonds to make proteins (remember ch 3 or so?)

· amino group NH2 of one aa is linked to carboxyl COOH of another aa

· tRNA made from tRNA genes on DNA

· aminoacyl-tRNA synthetase enzyme links aa’s to tRNA, using ATP energy.

· aminoacyl tRNA = tRNA with its joined amino acid

· tRNA holds the aa’s in position to make the peptide bond of the protein

tRNA has to

1. recognize by specific aminoacyl synthetase enzymes, to add the proper amino acid (aa)

2. have region to attach to the amino acid

3. recognize ribosomes

4. have ANTICODON, specific complementary binding sequence for the right mRNA codon

· tRNA = about 70 nucleotides

· complementary base pairing --> folds in tRNA sequence (Fig 12-7)

· amino acid binds at 3’ end (taller open end)

· aa carboxyl group binds to tRNA, so aa amino group can be in peptide bond

· bottom loop of tRNA has the anticodon region to complement mRNA

Ribosomes: cell organelle that translates mRNA into proteins (fig 12-8a)

• 2 subunits, each with protein and rRNA
• rRNA = structure and catalyst
• mRNA fits into groove between small and large subunit
• 2 depressions= A and P
• A = aminoacyl tRNA binds
• P = polypeptide chain fits here

Steps

1. Initiation

• Needs initiation factor proteins
• Load initiation tRNA onto the small subunit (AUG codon = tRNA for methionine) --> initiation complex
• Complex binds to mRNA at ribosome recognition sequences near 5’ end, which are upstream of coding sequences
• Initiator mRNA then lined up with the AUG codon
• Large subunit binds --> complete ribosome

2. Elongation: adding more amino acids with tRNA (fig 12-10)

• P site has initiator tRNA
• The next tRNA, as determined by the mRNA sequence, is added to the A spot
• Base pairing between the mRNA codon and the tRNA anticodon
• Energy from GTP
• Amino group on tRNA aa binds with carboxyl group of the amino acid in the P spot
• Peptide bond forms
• Amino acid at P site is released from its tRNA (spontaneous with ribozyme enzyme), and the naked tRNA leaves
• The polypeptide chain is now attached to the tRNA in the A spot
• Translocation: the complex moves down one codon: the A tRNA moves to the P spot. Energy from GTP.
• According to the bases in the next codon, the next tRNA binds to the A spot, and everything repeats
• Protein synthesis goes from the amino end to the carboxyl end of the growing chain
• MRNA is translated 5’ to 3’

3. Terminated by release factors that recognize stop codon, ribosome separates

Polyribosome aka polysome = mRNA molecule bound to cluster of ribosome

Prokaryotes couple transcription and translation.

• Make protein as mRNA is being made
• Half-life = 2mins; degrades quickly

Eukaryote mRNA needs post-transcriptional modification and processing

• Chromosomes in nucleus; protein synthesis in cytoplasm
• When mRNA about 20-30 nucleotides long, 5’ end is capped with 7-methylguanylate
• Cap maybe stability: half-life 30 min – 24 hours
• 3’ end gets polyA tail, may help export out of nucleus
• Introns = intervening sequences that are cut out (excised) from mRNA; The remaining exons (like the gas) are joined together (ligated) to make the functional mRNA (fig 12-12)
• Precursor mRNA pre-mRNA = large transcript with both introns and exons, transcribed from gene
• SnRNP (snerps) = small nuclear ribonucleoprotein complexes bind to introns, catalyze excise and splicing

Genetic code = nonoverlapping triplets of bases.

• Read one triplet (3 bases) at a time
• Fixed starting point sets the reading frame (123123123)
• Change reading frame means the nucleotide bases won’t be read in the right order ie. A triplet can become 231
• Nirenberg and Matthaei assigned specific triplets to specific amino acids
• UAA, UGA, UAG no amino acids, they are stop codons
• Table 12-1. Cool, huh, especially since they are universal. Don’t memorize them.
• Genetic code is bases in mRNA, which are complementary to bases in DNA and bases in tRNA
• Redundant: 64 possible codes, with 20 amino acids. Most amino acids have more than one triplet that codes for the aa
• Wobble hypothesis: some tRNA anticodons associate with more than one mRNA codon; the 5’ base of the anticodon can hydrogen bond with more than one kind of base in the 3’ position/ Crick

Gene = nucleotide sequence that carries the info needed to produce a specific RNA or protein product

• Coding and noncoding sequences

Mutations = changes in the nucleotide sequence of DNA

• Certain enzymes repair DNA changes
• Changes in DNA are copied to send to daughter cells
• Mutation = diversity, variation needed for evolution to happen in a species !

Base substitution mutation: change in 1 pair of nucleotides

• error in base pairing
• --> altered mRNA --> protein different by 1 amino acid

Missense mutation: replace one amino acids with another because 1 base is changed.

• Can drastically change enzyme active site
• Silent: not detectable, no obvious effect

Nonsense mutation: convert amino acid codon to termination codon

• Product usually won’t work
• Missing what would follow after the stop codon

Frameshift mutation: one or 2 nucleotide pairs inserted into or deleted from molecule

• Changes reading frame 123123--> 23123
• Codons downstream of insertion site have new sequence of amino acids
• New polypeptide made
• Stop codons within short distance of mutation
• Lose enzyme activity

Change chromosome structure (ch 13, 15, 16)

Transposons = DNA segment that is capable of moving from one chromosome to another or to different sites within the same chromosome; aka transposable element or mobile genetic element

• Disrupt active gene
• Turn on inactive gene
• Retroviruses copy the viral RNA into DNA, then DNA is inserted into host DNA
• If inserted in control region of a gene, the gene can be activated too highly
• Cancer causing viruses insert active DNA into regions controlling cell division.( Focus, p 282)

Hot Spots = regions of DNA much more likely to undergo mutations

• Sequence of repeated nucleotides
• Mutate DNA polymerase --> less accurate in copying DNA, so more mutations

Mutagen = agent that causes mutation

• Radiation: UV, xrays, etc
• Chemicals that change specific DNA bases, so base pairing messes up

Somatic cell mutations not passed on to offspring. Can lead to Cancer, though (carcinogen = cancer causing agent)

Mutations in sex cells (egg or sperm) are passed to offspring.