Chapter 15: Genes and How They Work
1. The Central Dogma (see handout)
a. Work of Crick after the Nobel Prize for DNA structure
b. Three basic principles that trace flow of gene encoded information
i. All DNA is copied from other DNA (replication)
ii. All RNA is copied from DNA (transcription)
iii. All proteins are copied from RNA (translation)
1. Three sequential RNA nucleotide bases specify one amino acid in
a protein chain (triplet code)
2. Genetic code is universal for all organisms
c. Two exceptions in retroviruses (RNA genetic material)
i. RNA replicates RNA
ii. RNA specifies DNA (reverse transcription)
d. Principle of collinearity
i. Sequence of deoxyribonucleotides (DNA)  transcription  Sequence of
Ribonucleotides (mRNA) Translation (protein synthesis) Sequence of
amino acids (protein)
2. Transcription: Synthesis of RNA
a. Process is well understood in Prokaryotes
b. mRNA is the intermediary molecule between the genetic code on DNA and
protein synthesis
c. RNA polymerase is complex with 5 subunits
i. 2 subunits bind regulatory proteins to DNA
ii. 1 subunit binds to DNA
iii. 1 subunit binds RNA nucleotides
iv. 1 subunit recognizes the promoter site and begins synthesis
d. Promoter Site
i. Binding site for RNA polymerase on DNA upstream from the gene
ii. Not Transcribed
iii. Specific sequences, ex: TATAAT, -10 upstream
iv. In bacteria, may be strong or weak, determines how frequent gene is
transcribed
e. Initiation – handout #297
i. RNA polymerase binds to promoter site and DNA unzips and separates 17
bps
ii. One side acts as a template at a time (both sides have genes) or antisense
strand
iii. Other side is the coding strand or sense strand (same of mRNA)
f. Elongation
i. RNA polymerase binds and no primer necessary
ii. Triphosphate ribonucleotides complementary base pair on template
strand (begins with ATP or GTP in prokaryotes)
iii. Uracil replaces thymine
iv. mRNA grows 5’ to 3’ (60 nucleotides/sec)
v. Transcription bubble forms (fig 15.9)
vi. As mRNA formed, DNA helix reforms, and mRNA leaves template
vii. No proofreading
g. Termination
i. At end of prokaryotic gene are “termination” sequences
ii. Causes formation of phosphodiester bonds to cease, mRNA to
disassociate, DNA rewind, RNA polymerase to be released
h. mRNA in prokaryotes may contain many genes –polycistronic, because related
genes are adjacent to each other
i. Translation may be continuous with transcription because no nuclear membrane
j. See Becker’s website
k. http://bcs.whfreeman.com/thelifewire/content/chp12/1202001.html
3. Transcription in Eukaryotes – basically same, except for the following differences
a. 3 RNA polymerases
i. Each recognizes different promoters
ii. Pol I transcribes rRNA
iii. Pol II transcribes mRNA, uses promoter “TATA box”
iv. Pol III transcribes tRNA
b. Initiation
i. Requires a complex of protein transcription factors to form an
initiation complex to help bind pol II – see handout #299
c. Elongation
i. Only one gene per mRNA transcript.
d. See Becker’s website
e. Posttranscriptional Modifications
i. Addition of 5’ cap
1. Modified guanosine nucleotide with methyl group: 7-methyl
guanosine
2. Added while transcription is still in progress
3. Allows passage through nuclear pores without degradation
4. Heads with initiation of translation on ribosome
ii. Addition of Poly-A tail at 3’ end
1. 200 Adenine nucleotides added at end of mRNA transcript
2. Prevents degradation of mRNA by enzymes
f. Eukaryotic Transcripts are Spliced/Edited – see handout #306
i. Only eukaryotic DNA has non-coding sequences of nucleotides called
introns (intervening sequences) within the coding sequences, exons,
(expressed sequences)
ii. In humans, only 1-1.5% genome is exons (expressed)
iii. Purpose of introns
1. Separate polypeptide chains in a protein
2. Promote crossing over because increase distance between genes,
promotes genetic variability
iv. Introns must be removed from mRNA transcript prior to translation
v. Splicing and Editing mRNA (RNA processing) – see handout #307
1. Remove introns from primary transcript of mRNA and splice
together exons to form mature mRNA
2. Use of snRNPs (small nuclear ribonuclearprotein) = snRNA +
protein
a. Occurs in nucleus before mRNA leaves for translation
b. Recognize junction between introns and exons
c. Base pairs with ends of intron
d. Clusters of snRNPs + proteins form spliceosome slides
over primary transcript, cuts out introns and splices
together remaining exons
3. Alternate Splicing – see page 349, fig 17.8 and pg 376
a. Many different but similar proteins can be synthesized from
the same primary mRNA transcript
b. Spliceosomes can remove or keep different introns and
exons to form various mature mRNA, therefore different
proteins.
4. How Genes Encode Information
a. Francis Crick’s experiments determined that a triplet code of 3 nucleotides
specifies each amino acid in protein
i. Four base pairs for 20 amino acids
ii. 41 = 4 codes if groups of 1 bp (not enough)
iii. 42 = 16 codes if groups of 2bp (not enough)
iv. 43 = 64 codes if groups of 3bp (extra codes – degenerate code, more than
one triplet per amino acid)
v. Triplet code of deoxyribonucleotides on DNA specifies a triplet code of
ribonucleotides on mRNA called a codon
1. DNA – triplet code of deoxyribonucleotide
2. mRNA – codons – three ribonucleotides complementary to DNA
3. tRNA – anti-codons – complementary to codons
b. 1961: Nierenberg, Matthei, Khorana broke the code (see table 15.1) (read
method in text)
c. Multiple codes for an amino acid allows for mutation without change in protein
i. Differ in third nucleotide at 3’ end.
ii. When anticodon on tRNA base pairs with the third base, it can wobble,
but still be translated into correct amino acid: Wobble hypothesis
d. Some tRNA have a base inosine which pairs with A, C, and G
e. 61 of the 64 codons code for amino acids
i. One start codon: AUG, codes for f-met *(formyl methionine), modified
amino acid
ii. Three stop codons: UGA, UAG, UAA (non-sense codons)
f. Genetic code is universal, except for mitochondrial DNA (some codes are
different)
g. Point Mutation – change in genetic code’s base sequence that causes a change in
amino acid sequence
5. Translation: The Synthesis of Proteins – see handout #292
a. Information contained in sequence of mRNA is translated into protein on
ribosome
b. Role of transfer RNA (tRNA) as “interpreter”
c.
d.
e.
f.
i. Recognizes the codon on mRNA
ii. Picks up the appropriate amino acid in cytoplasm
Structure of tRNA and its function
i. Example of structure/function, specificity, recognition themes!
ii. Single stranded, folds upon itself
iii. Four recognition sites
Amino Acid Activation Enzymes (aminoacyl-tRNA synthetases) – see
handout #302
i. Attach specific amino acids to tRNA
ii. Each of the 20 amino acids has a specific enzyme to attach it to tRNA
(some have multiple RNAs why?)
iii. Mechanism powered by ATP
iv. Activating enzyme has two active sites:
1. For the amino acid (trp)
2. For tRNA
v. Using enzyme from ATP, a covalent bond is formed between the carboxyl
group of amino acid and –OH group on tRNA.
vi. When the “amino acid tRNA” complex is formed, it is released from the
enzyme. The amino acid is “activated”
Role of Ribosomes – Site of Protein Synthesis (translation)
i. Coordinated the coupling of tRNA and mRNA
ii. Prokaryotic ribosomes – (30s/50s) subunits
Eukaryotic ribosomes – (40s/60s) subunits
iii. Large subunit – Three binding sites
1. P-site: holds tRNA with growing PolyPeptide chain
2. A-site: holds tRNA with next Amino Acid
3. E-Site: tRNA exits
iv. Small subunit
1. Binding site for mRNA
The Process of Protein Synthesis/Translation of mRNA in prokaryotes
i. Three stages: All stages require enzymes, protein “factors,” and
energy from GTP
1. Chain initiation
2. Chain elongation
3. Chain termination
ii. Chain initiation: formation of initiation complex (see handout #303)
1. mRNA attaches to small subunit of ribosome with leader sequence
complementary to rRNA
2. Initiation factors align tRNA with f-met with AUG on mRNA
(start codon)
3. Large subunit attaches to small subunit. E-site is empty, P-site
holds initiator tRNA with f-met, A-site is empty, forming a
“reading frame” for the next codon (3 bases)
iii. Eukaryotes differ
1. Use methionine instead of f-methionine
2. Initiation complex involves more initiation factors.
iv. Chain Elongation – Translocation – see handout #304
1. Ribosome moves along mRNA, 5’ to 3’, by 3 nucleotides, helped
by elongation factors & GTP
a. Next tRNA moves into the A-site, recognizing the codon in
reading frame, base pairs with its anti-codon
b. Peptidyl transferase in large subunit forms peptide bond
between the amino acids at the P and A sites. Amino acid at
P site is released from its tRNA
c. tRNA at P-site moves into the E-site and is released. The
tRNA at the A-site moves into the P-site with growing
polypeptide
v. Termination – see handout #305
1. When one of 3 STOP* codons appears in A-site, Release factor,
from large sub-unit, binds to it, not tRNA! *UAA, UAG, UGA
2. Peptidyl transferase adds water to end of polypeptide chain,
releasing it from last tRNA
3. Large and small subunit disassociates, releasing mRNA and tRNA
vi. Overview of Eukaryotic Gene Expression – see handout #308
vii. Differences between prokaryotic and eukaryotic gene expression
1. Transcription occurs in nucleus and translation occurs in ribosomes
in eukaryotes; transcription and translation in prokaryotes occur in
cytoplasm
2. There is no introns and exons in prokaryotes; eukaryotes have
intron/exon splicing
3. Prokaryotes don’t have 5’ cap or 3’ poly-A tail; eukaryotes have
them
4. Prokaryotes start with F-met; Eukaryotes start with met
5. Prokaryotes have 30s/50s ribosomes; eukaryotes have 40s/60s
ribosomes
6. In prokaryotes, mRNA is polycistone, while in eukaryotes, only
one per mRNA
7. Eukaryotic ribosomes larger
8. Prokaryotes only have one RNA polymerase, Eukaryotes have
three different RNA polymerases
9. More initiation factors in eukaryotes