Chapter 12: RNA and Protein Synthesis Gene Expression – How DNA affects Phenotype DNA  proteins  phenotype

2 steps DNA  mRNA Translation mRNA  protein

Nuclear envelope DNA TRANSCRIPTION Pre-mRNA mRNA TRANSLATION Ribosome Fig. 17-3b-3 Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING mRNA Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

Gene 2 Gene 1 Gene 3 DNA template strand mRNA Codon TRANSLATION Fig. 17-4 Gene 2 DNA molecule Gene 1 Gene 3 DNA template strand TRANSCRIPTION Figure 17.4 The triplet code mRNA Codon TRANSLATION Protein Amino acid

RNA – ribonucleic acid Single stranded nucleotides Ribose Phosphate AUCG U = Uracil

RNA Transcription (DNA template  mRNA) 3 types Ribosomal RNA (rRNA) Transfer RNA (tRNA) Messenger RNA (mRNA) Made from DNA – DNA-dependent RNA polymerases Make RNA from DNA in 5’ 3’ direction, DNA read 3’5’ DNA template and new RNA are antiparallel

Upstream – toward 5’ of mRNA OR 3’ of DNA Downstream – toward 3’ of RNA OR 5’ of DNA

Transcription 1. RNA polymerase binds to DNA at Promoter Promoter not transcribed RNA polymerase passes promoter; begins transcribing DNA No primer required 2. RNA nucleotides added to 3’ end of RNA 1st RNA (5’ end) keeps triphosphate RNA nucleotides added lose 2 P and 3rd P becomes part of sugar-phosphate backbone Last RNA nucleotide – exposed 3’ OH

Transcription 3. Termination Stop sequence at end of gene

Several transcription factors must bind to the DNA before RNA Fig. 17-8 1 A eukaryotic promoter includes a TATA box Promoter Template 5 3 3 5 TATA box Start point Template DNA strand 2 Several transcription factors must bind to the DNA before RNA polymerase II can do so. Transcription factors 5 3 3 5 3 Additional transcription factors bind to the DNA along with RNA polymerase II, forming the transcription initiation complex. Figure 17.8 The initiation of transcription at a eukaryotic promoter RNA polymerase II Transcription factors 5 3 3 5 5 RNA transcript Transcription initiation complex

Completed RNA transcript Fig. 17-7a-4 Promoter Transcription unit 5 3 3 5 DNA Start point RNA polymerase 1 Initiation 5 3 3 5 RNA transcript Template strand of DNA Unwound DNA 2 Elongation Rewound DNA 5 3 3 3 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination 5 RNA transcript 3 Termination 5 3 3 5 5 3 Completed RNA transcript

Nontemplate Elongation strand of DNA RNA nucleotides RNA polymerase 3 Fig. 17-7b Elongation Nontemplate strand of DNA RNA nucleotides RNA polymerase 3 3 end 5 Figure 17.7 The stages of transcription: initiation, elongation, and termination 5 Direction of transcription (“downstream”) Template strand of DNA Newly made RNA

Resulting mRNA Leader sequence – Coding sequence – Noncoding Made because RNA polymerase starts transcription well upstream of coding sequence Coding sequence – Codes for proteins Termination or stop codon End of coding sequence UAA, UGA, UAG Don’t code for AA; specify end of protein Followed by noncoding 3’ trailing sequences

Transcription

Posttranscriptional modification and processing Precursor mRNA = original mRNA transcript (pre-mRNA) Begins – RNA transcript is 20-30 nucleotides long Enzymes add cap to 5’ end of mRNA Need cap for eukaryotic ribosome to bind May protect from degradation

Protein-coding segment Polyadenylation signal 5 3 Fig. 17-9 Protein-coding segment Polyadenylation signal 5 3 G P P P AAUAAA AAA … AAA 5 Cap 5 UTR Start codon Stop codon 3 UTR Poly-A tail Figure 17.9 RNA processing: addition of the 5 cap and poly-A tail

Polyadenylated (poly-A) tail gets added 3’ end When transcript complete, enzymes in nucleus recognize polyadenylation signal and cut mRNA at that site 100-250 adenine nucleotides are added by enzymes to 3’ end May help Export mRNA from nucleus, fight degradation, make translation initiation more efficient

Take out noncoding sequences Interrupted coding sequences = long sequences of bases in the protein-coding sequences of the gene that do not code for AA in the final protein product INTRONS (noncoding regions) EXONS – (expressed sequences) – part of the protein-coding sequence Introns are removed and splice exons together  continuous coding sequence

Small nuclear ribonucleoprotein complexes (snRNPs) – bind to introns and catalyze the excision and splicing reactions

exons spliced together Coding segment Fig. 17-10 5 Exon Intron Exon Intron Exon 3 Pre-mRNA 5 Cap Poly-A tail 1 30 31 104 105 146 Introns cut out and exons spliced together Coding segment mRNA 5 Cap Poly-A tail 1 146 Figure 17.10 RNA processing: RNA splicing 5 UTR 3 UTR

RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Fig. 17-11-3 RNA transcript (pre-mRNA) 5 Exon 1 Intron Exon 2 Protein Other proteins snRNA snRNPs Spliceosome 5 Figure 17.11 The roles of snRNPs and spliceosomes in pre-mRNA splicing Spliceosome components Cut-out intron mRNA 5 Exon 1 Exon 2

mRNA processing

Amino acids tRNA with amino acid attached Ribosome tRNA Anticodon 5 Fig. 17-13 Amino acids Polypeptide tRNA with amino acid attached Ribosome Trp Phe Gly Figure 17.13 Translation: the basic concept tRNA Anticodon 5 Codons 3 mRNA

Translation: mRNA  AA (protein) Codon – sequence of 3 consecutive bases in mRNA (triplet code); specify 1 AA Transfer RNA (tRNA) – connect AA and mRNA; link with specific AA Anticodon – sequence of 3 bases on tRNA; H bonds with mRNA codon by base-pairing rules

Aminoacyl-tRNA synthetase – enzyme that links amino acids to tRNAs Make aminoacyl-tRNAs (can bind to mRNA)

Aminoacyl-tRNA Amino acid synthetase (enzyme) tRNA Aminoacyl-tRNA Fig. 17-15-4 Aminoacyl-tRNA synthetase (enzyme) Amino acid P P P Adenosine ATP P Adenosine tRNA P P i Aminoacyl-tRNA synthetase P i P i tRNA Figure 17.15 An aminoacyl-tRNA synthetase joining a specific amino acid to a tRNA P Adenosine AMP Computer model Aminoacyl-tRNA (“charged tRNA”)

Properties of tRNA 1. anticodon – 3 base triplet complementary to mRNA codon 2. must be recognized by specific aminoacyl-tRNA synthetase that adds correct AA 3. must have region that serves as attachment site for specific AA specified by anticodon 4. must be recognized by ribosomes

tRNA Gets folded on itself (base-pairing within tRNA)  3+ loops (unpaired bases) 1 of the loops has anticodon 3’ end – AA binding site Carboxyl of AA binds to OH tRNA at 3’ end, leaving amino group on AA to make peptide bond

(a) Two-dimensional structure Fig. 17-14a 3 Amino acid attachment site 5 Hydrogen bonds Figure 17.14 The structure of transfer RNA (tRNA) Anticodon (a) Two-dimensional structure

Translation – At Ribosomes Made of 2 different subunits (proteins and ribosomal RNA) rRNA – no transfer of info, has catalytic functions Attach to 1 end of mRNA and travel along it, allowing tRNAs to attach in sequence to mRNA codons

Ribosomes mRNA fits in groove between 2 subunits Holds mRNA, aminoacyl tRNA and growing polypeptide chain tRNAs attach to A and P binding sites A site – new AA dock; AA form bond with polypeptide chain and tRNA moves to P site

(b) Schematic model showing binding sites Fig. 17-16b P site (Peptidyl-tRNA binding site) A site (Aminoacyl- tRNA binding site) E site (Exit site) E P A Large subunit mRNA binding site Small subunit (b) Schematic model showing binding sites Growing polypeptide Amino end Next amino acid to be added to polypeptide chain Figure 17.16 The anatomy of a functioning ribosome E tRNA mRNA 3 Codons 5 (c) Schematic model with mRNA and tRNA

3 stages of Translation Initiation, repeating cycles of elongation, termination

Initiation Initiation factors (proteins) attach to small subunit, allowing it to bind to a special initiator tRNA Initiator tRNA is loaded onto small subunit, making initiation complex Initiation complex binds to special ribosome-recognition sequences upstream of coding sequences on mRNA (near 5’ end) Initiation complex moves along mRNA until it reaches an initiator codon = AUG

Anticodon of initiator tRNA binds to initiation codon of mRNA Large subunit attaches to complex  completed ribosome

Translation initiation complex Fig. 17-17 Large ribosomal subunit 3 U C 5 A P site Met 5 A Met U G 3 Initiator tRNA GTP GDP E A mRNA 5 5 3 3 Start codon Figure 17.17 The initiation of translation Small ribosomal subunit mRNA binding site Translation initiation complex

Elongation Addition of AA to A site by base pairing of anticodon w/ codon Ribosome moves in 3’ direction along mRNA Needs energy from GTP Peptidyl transferase – ribozyme – rRNA component of large subunit AA at P site released from its tRNA (in P site) Peptidyl transferase attaches this AA to aminoacyl-tRNA at A site Peptide bond formed – translocation – chain moves to P site, leaving A site open GTP for bond, none for transferase

GDP GDP Amino end of polypeptide E 3 mRNA Ribosome ready for Fig. 17-18-4 Amino end of polypeptide E 3 mRNA Ribosome ready for next aminoacyl tRNA P site A site 5 GTP GDP E E P A P A Figure 17.18 The elongation cycle of translation GDP GTP E P A

Termination “Release factors” stop translation Recognize termination (stop) codons Release newly-made protein, mRNA and last tRNA, causing ribosome to dissociate

Release factor Free polypeptide 5 3 3 3 2 5 5 Stop codon Fig. 17-19-3 Release factor Free polypeptide 5 3 3 3 2 5 5 GTP Stop codon (UAG, UAA, or UGA) 2 GDP Figure 17.19 The termination of translation

Translation

Protein Synthesis: Eukaryotes vs. Prokaryotes mRNA is translated as it is being transcribed from DNA (no nucleus to exit) mRNA used immediately, no further processing mRNA must be transported to cytoplasm before translation Original mRNA transcript must be modified before leaving the nucleus

Fig. 17-3 DNA TRANSCRIPTION mRNA Ribosome TRANSLATION Polypeptide (a) Bacterial cell Nuclear envelope DNA TRANSCRIPTION Pre-mRNA RNA PROCESSING Figure 17.3 Overview: the roles of transcription and translation in the flow of genetic information mRNA TRANSLATION Ribosome Polypeptide (b) Eukaryotic cell

Special features of the Genetic Code 3 letter combos from 4 bases – specify 64 AA Nirenberg and Matthaei Experimented to determine which AA were coded for by specific mRNA codons Ex: UUUUUUUUU… - only found phenylalanine so UUU = phenylalanine Found 3 stop codons – specified no AA UAA, UGA, UAG

First mRNA base (5 end of codon) Third mRNA base (3 end of codon) Fig. 17-5 Second mRNA base First mRNA base (5 end of codon) Third mRNA base (3 end of codon) Figure 17.5 The dictionary of the genetic code

The Genetic Code is Universal All organisms Few coding exceptions Protozoans – UAA, UGA for glutamine, instead of stop Mitochondria - own DNA

Wobble Hypothesis 61 codons, but only 40 different tRNAs  tRNA can pair w/ 1+ codon Francis Crick 3rd nucleotide of tRNA anticodon (5’ end) may be capable of forming H bonds w/ more than 1 kind of 3rd nucleotide (3’ end) of codon

Reverse? Howard Temin – proposed DNA provirus formed as intermediate in replication of RNA tumor viruses RNA-directed DNA polymerase (Reverse transcriptase) – made DNA from RNA template Retroviruses HIV

Mutations Changes in nucleotide sequence of DNA Spontaneously during DNA replication, mitosis, meiosis, or because of mutagens Low rate of occurrence because of cell’s repair mechanisms Provide diversity of genes Variation evolution Copied as normal, no greater chance of further mutation (normally)

Base substitution mutation Simplest 1 pair nucleotides changes From errors in base pairing during replication Affects transcribed mRNA and polypeptide

Missense mutations Base substitutions that result in the replacement of 1 AA by another Wide range of effects Enzyme activity “silent” – substituted w/ closely related AA, effects undetectable

Nonsense mutations Base substitution that converts an AA-specifying codon to a termination codon Usually destroys function of gene product

Frameshift mutations 1 or 2 nucleotide pairs are inserted or deleted from the molecule, causing an alteration of the reading frame Codons downstream now specify entirely new sequence of AA Different effects depends where it happens Entirely new polypeptide Stop codon w/in short distance of mutation Loss enzyme activity (disastrous)

Figure 17.23 Types of point mutations Wild-type DNA template strand 3 5 5 3 mRNA 5 3 Protein Stop Amino end Carboxyl end A instead of G Extra A 3 5 3 5 5 3 5 3 U instead of C Extra U 5 3 5 3 Stop Stop Silent (no effect on amino acid sequence) Frameshift causing immediate nonsense (1 base-pair insertion) T instead of C missing 3 5 3 5 5 3 5 3 A instead of G missing 5 3 5 3 Stop Figure 17.23 Types of point mutations Missense Frameshift causing extensive missense (1 base-pair deletion) A instead of T missing 3 5 3 5 5 3 5 3 U instead of A missing 5 3 5 3 Stop Stop Nonsense No frameshift, but one amino acid missing (3 base-pair deletion) (a) Base-pair substitution (b) Base-pair insertion or deletion

Jumping Genes (mobile genetic elements, transposable elements, transposons) DNA sequence “jumps” to middle of gene, disrupting gene functions and/or activation previously inactive genes Genes spontaneously turned on or off Barbara McClintock – 1950s Require transposase enzyme

Mutagens Mutagens Carcinogens Agents that cause mutations Radiation – X, gamma, cosmic, UV rays; chemicals Carcinogens Cause cancer

Fig. 17-25 DNA TRANSCRIPTION 3 Poly-A RNA polymerase 5 RNA transcript RNA PROCESSING Exon RNA transcript (pre-mRNA) Intron Aminoacyl-tRNA synthetase Poly-A NUCLEUS Amino acid AMINO ACID ACTIVATION CYTOPLASM tRNA mRNA Growing polypeptide Cap 3 A Activated amino acid Poly-A P Ribosomal subunits Figure 17.25 A summary of transcription and translation in a eukaryotic cell E Cap 5 TRANSLATION E A Anticodon Codon Ribosome

Fig. 17-UN8