What is a gene?

1
What is a gene?

• A sequence of DNA nucleotides that specifies the
  primary structure of a polypeptide chain (tells
  the cell how to make it)
• Genes-made of nucleotides
• Proteins-made of amino acids
• How does a nucleotide code (in the nucleus)
  specify an amino acid sequence (in the cytoplasm)?

2
The Central Dogma

• DNA is transcribed into RNA-characteristics of
  RNA
• RNA is translated into protein
• Advantages
• Exceptions

3
LE 17-4
Gene 2
DNA molecule
Gene 1
Gene 3
5
3
DNA strand (template)
TRANSCRIPTION
3
5
mRNA
Codon
TRANSLATION
Protein
Amino acid
4
The Genetic code-characteristics

• Triplet (3 nucleotidescodoninfo for a specific
  amino acid)64 different codons (3 are stop
  codons)
• Universal
• Redundant (61 codons-20 amino acids)-variability
  in third nucleotide of codon. Advantages of a
  redundant code?
• Non-overlapping
• Exceptions (ciliates mito/chloroplasts)

5
LE 17-5
Second mRNA base
First mRNA base (5 end)
Third mRNA base (3 end)
6
Figure 17-06
7
Gene Expression

• If a gene is transcribed and the m-rna is
  translated (the gene is expressed) a protein is
  made. This often changes the phenotype of the
  cell that produces the protein.
• Differential gene expression is involved in
  embryonic development and cell specialization.
• Totipotency-each cell has the genetic information
  for an entire organism.
• Differential gene expression results in cell
  specialization (differentiation)
• Hormones often play a role in gene expression

8
Transcription

• The first step in gene expression
• Takes place in the nucleus
• Requirements
• A. RNA nucleotides
• B. DNA template (gene)
• C. Enzymes (RNA polymerase)
• Only one of the two DNA strands is copied
  (template strand)

9
LE 17-7a-1
Promoter
Transcription unit
5
3
3
5
DNA
Start point
RNA polymerase
10
LE 17-7a-2
Promoter
Transcription unit
5
3
3
5
DNA
Start point
RNA polymerase
Initiation
3
5
5
3
Template strand of DNA
RNA tran- script
Unwound DNA
11
LE 17-7a-3
Promoter
Transcription unit
5
3
3
5
DNA
Start point
RNA polymerase
Initiation
3
5
5
3
Template strand of DNA
RNA tran- script
Unwound DNA
Elongation
Rewound DNA
3
5
3
5
3
5
RNA transcript
12
LE 17-7a-4
Promoter
Transcription unit
5
3
3
5
DNA
Start point
RNA polymerase
Initiation
5
3
5
3
Template strand of DNA
RNA tran- script
Unwound DNA
Elongation
Rewound DNA
5
3
3
3
5
5
RNA transcript
Termination
3
5
3
5
5
3
Completed RNA transcript
13
LE 17-7b
Elongation
Non-template strand of DNA
RNA nucleotides
RNA polymerase
3
3 end
5
Direction of transcription (downstream)
5
Template strand of DNA
Newly made RNA
14
LE 17-8
Eukaryotic promoters
Promoter
5
3
3
5
TATA box
Start point
Template DNA strand
Several transcription factors
Transcription factors
5
3
3
5
Additional transcription factors
RNA polymerase II
Transcription factors
5
3
5
3
5
RNA transcript
Transcription initiation complex
15
Transcription-some important details

• Rate-30-60 nucleotides/second
• RNA polymerase (Many forms in eucaryotes, 3 basic
  types in bacteria type I transcribes r-rna, type
  II-mrna, types III-trna)
• Promotors-(approximately 100 nucleotides)-strong
  and weak promotors
• Eukaryotes-transcription factors needed to help
  RNA polymerase to bind to TATA box (region of
  promotor 25 nucleotides upstream from initiation
  site).

16
RNA products of transcription
17
Recent discoveries indicate that a large part of
the eukaryotic genome is non-coding RNA

• R-rna and T-trna are examples
• Small rna (micro rna and small interfering
  rna)-play a crucial role in the regulation of
  gene expression involving both transcription and
  translation
• Rna interference
• Well talk about regulation of gene expression in
  Chapter 18.

18
Ribosomal RNA and ribosomes

• R-rna one of two important components of
  ribosomes (other is protein-some of the proteins
  are enzymes). 60 r-rna 40 protein.
• Ribosomes consist of 2 subunits
• Ribosomes needed to translate proteins
• workbench of protein synthesis
• Position t-rna (which is attached to a specific
  amino acid) on the codon of a m-rna
• Result is the synthesis of a protein (whose amino
  acid sequence is specified by the m-rna which is
  transcribed from a gene)

19
LE 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
Schematic model showing binding sites
20
LE 17-16a
Exit tunnel
Growing polypeptide
tRNA molecules
Large subunit
E
P
A
Small subunit
5
3
mRNA
Computer model of functioning ribosome
21
Ribosomal rna processing
22
T-rna

• Single polynucleotide chain folded into a complex
  3-D shape (inter-chain H bonding). 75-80
  nucleotides in length
• Binds a specific amino acid (involvement of
  amino-acyl-trna-synthetase
• Attaches to a specific m-rna codon via its
  anticodon
• How many different t-rnas are there? 61?
  Actually only 45 (wobble)

23
LE 17-14a
3
Amino acid attachment site
5
Hydrogen bonds
Anticodon
Two-dimensional structure
Amino acid attachment site
5
3
Hydrogen bonds
3
5
Anticodon
Anticodon
Three-dimensional structure
Symbol used in this book
24
Charging t-rna with its specific amino acid

• charging enzyme-amino acyl t-rna synthetase (20
  different enzymes)
• Requires ATP

25
LE 17-15
Amino acid
Aminoacyl-tRNA synthetase (enzyme)
Pyrophosphate
Phosphates
tRNA
AMP
Aminoacyl tRNA (an activated amino acid)
26
Messenger Rna (m-rna)

• Contains the information for the primary sequence
  of a polypeptide chain
• Consists of codons
• Binds to ribosomes
• T-rna binds to m-rna (codon/anticodon)

27
LE 17-13
Amino acids
Polypeptide
tRNA with amino acid attached
Ribosome
tRNA
Anticodon
Codons
5
3
mRNA
28
Translation

• Codons (m-rna) read by ribosomes/t-rna
• Polypeptide chain produced
• 3 steps in translation-
• A. initiation
• B. elongation
• C. termination
• Translation is a process that consumes a
  tremendous amount of energy (ATP and GTP)

29
LE 17-16c
Amino end
Growing polypeptide
Next amino acid to be added to polypeptide chain
E
tRNA
mRNA
3
Codons
5
Schematic model with mRNA and tRNA
30
Translation-Initiation

• Initiation codon is AUG
• T-rna that bonds to AUG has an anticodon UAC-this
  carries the amino acid methionine
• Requires a GTP molecule
• Requires proteins called initiation factors.

31
LE 17-17
Large ribosomal subunit
P site
Met
Met
Initiator tRNA
GTP
GDP
A
E
mRNA
5
5
3
3
Start codon
Small ribosomal subunit
mRNA binding site
Translation initiation complex
32
Translation-Elongation

• The elongation cycle takes about 60 milliseconds
• During elongation, one m-rna codon is read and
  then the ribosomes moves down the message to the
  next codon.
• Binding of incoming t-rna to the A site of the
  ribosome requires a GTP
• Translocation-requires a GTP

33
LE 17-18
Amino end of polypeptide
E
3
mRNA
P site
A site
Ribosome ready for next aminoacyl tRNA
5
2
GTP
2 GDP
E
E
P
A
P
A
GDP
GTP
E
P
A
34
Translation-Termination

• When the ribosome reaches a termination codon, it
  causes the m-rna/ribosome complex to separate
• No t-rna binds to the termination codon.
• Release factors
• Newly made polypeptide chain is released (folds
  into its characteristic 3-D shape)

35
LE 17-19
Release factor
Free polypeptide
5
3
3
3
5
5
Stop codon (UAG, UAA, or UGA)
When a ribosome reaches a stop codon on mRNA, the
A site of the ribosome accepts a protein called a
release factor instead of tRNA.
The release factor hydrolyzes the bond between
the tRNA in the P site and the last amino acid of
the polypeptide chain. The polypeptide is thus
freed from the ribosome.
The two ribosomal subunits and the other
components of the assembly dissociate.
36
Summary of energy demands for protein synthesis

• A rough estimate is that for every amino acid
  incorporated into a polypeptide chain, 3 ATP/GTP
  are consumed
• Charging the amino acid (1 ATP)
• Binding of incoming t-rna into the A site (1 GTP)
• Translocation (1 GTP)
• So a small protein (120 amino acids in length)
  would cost the cell 360 ATP/GTP to make (the
  equivalent of 12 glucose molecules going through
  aerobic cell respiration)

37
Polyribosomes

• A single ribosome can translate an average-sized
  polypeptide in about 1 minute
• Several ribosomes can translate the same message
  one after the other.
• Increases the efficiency of protein production

38
LE 17-20a
Completed polypeptide
Growing polypeptides
Incoming ribosomal subunits
Polyribosome
Start of mRNA (5 end)
End of mRNA (3 end)
An mRNA molecule is generally translated
simultaneously by several ribosomes in clusters
called polyribosomes.
39
LE 17-20b
Ribosomes
mRNA
m
0.1 m
This micrograph shows a large polyribosome in a
prokaryotic cell (TEM).
40
M-rna modifications

• Eukaryotic M-rna is modified extensively after
  transcription (while its still in the nucleus)
• These modifications include
• A.Polyadenylation-added to 3 end of m-rna
• B. 5 cap
• C. Intron removal

41
M-RNA modifications

• Poly A tail
• A. added to the 3 end of the m-rna
• B.30-200 Adenine nucleotides
• C. roles-regulation of transport of m-rna out of
  the nucleus regulation of degradation of m-rna
  in the cytoplasm helps m-rna attach to small
  ribosomal subunit

42
M-RNA modifications (continued)

• 5 cap
• A. Modified guanine nucleotide stuck onto 5 end
  of m-rna
• B. Roles- positioning of m-rna on small ribosomal
  subunit in initiation protects m-rna from
  degradation

43
LE 17-9
Protein-coding segment
Polyadenylation signal
5
Start codon
Stop codon
3
UTR
5
5
Cap
UTR
Poly-A tail
44
Introns

• Many eukaryotic genes have nucleotide sequences
  that dont code for amino acids (Introns)
• Introns separate coding sequences (exons). Split
  genes
• Introns must be removed from the m-rna before it
  is translated (introns have nucleotide sequences
  that indicate splicing sites)
• Splicesomes are molecular machines that remove
  introns from m-rna

45
LE 17-11-1
RNA transcript (pre-mRNA)
5
Intron
Exon 1
Exon 2
Protein
Other proteins
snRNA
snRNPs
Spliceosome
46
LE 17-11-2
Spliceosome
5
Spliceosome components
Cut-out intron
mRNA
5
Exon 1
Exon 2
47
Significance of introns

• Why would chromosomes carry around extra DNA that
  isnt used in the final m-rna?
• Expensive to maintain (energy).
• Splicing out introns is a risky business (what if
  its done incorrectly)
• With these disadvantages, there must be an
  advantage or natural selection would not favor
  this arrangement

48
Benefits of Introns

• Evolution of protein diversity
• One gene can be alternatively spliced in a number
  of different ways to form several different types
  of m-rna (alternative splicing)
• Human antibody genes-about 500 genes can code for
  billions of different antibody molecules because
  of alternative splicing.

49
LE 17-12
Gene
DNA
Exon 1
Intron
Exon 2
Intron
Exon 3
Transcription
RNA processing
Translation
Domain 3
Domain 2
Domain 1
Polypeptide
50
Summary of Transcription and Translation
51
Mutation

• An alteration in the nucleotide sequence of a DNA
  molecule (chromosome)
• Chromosomal mutations (duplications deletions
  inversions)
• Point mutations-alterations of one or a few
  nucleotides in a gene

52
Point mutations

• Spontaneous mutations
• Induced mutations
• Consequences of mutations-
• A. no effect-silent mutations
• B. harmful mutations-(may be lethal)
• C. beneficial mutations (rare)

53
Spontaneous mutations

• Base pairing errors why arent they corrected by
  DNA repair enzymes?
• Effects
• A. no effect-silent mutation (redundancy of
  genetic code alteration of a non-critical amino
  acid)
• B. Positive effect-rare
• C. negative effect-missense mutations nonsense
  mutations

54
LE 17-24a
Wild-type
mRNA
3
5
Protein
Stop
Amino end
Carboxyl end
55
LE 17-24b
Base-pair substitution
No effect on amino acid sequence
U instead of C
Stop
Missense
A instead of G
Stop
Nonsense
U instead of A
Stop
56
Sickle cell anemia

• Results of a spontaneous missense mutation
• Result-altered hemoglobin molecule
• Effect-Depends on the environmental conditions
  and number of copies of the defective gene you
  inherited.

57
LE 17-23
Wild-type hemoglobin DNA
Mutant hemoglobin DNA
3
5
5
3
mRNA
mRNA
5
3
3
5
Normal hemoglobin
Sickle-cell hemoglobin
58
(No Transcript)
59
(No Transcript)
60
(No Transcript)
61
Induced mutations

• Caused by environmental damage
• Radiation (UV)- T-T dimers excision repair
  enzymes xerdoerma pigmentosa
• Chemicals-Common result-base pair addition or
  deletion
• Result of addition or deletion (frame shift
  mutation)-missense or nonsense
• Worst scenario-addition/deletion of 1 or 2
  nucleotides at the beginning of a gene

62
LE 17-25
Wild type
mRNA
5
3
Protein
Stop
Carboxyl end
Amino end
Base-pair insertion or deletion
Frameshift causing immediate nonsense
Extra U
Stop
Frameshift causing extensive missense
Missing
Insertion or deletion of 3 nucleotides no
frameshift but extra or missing amino acid
Missing
Stop
63
Mutations and Cancer

• Many mutations make cells cancerous
• 90 of known carcinogens are mutagens
• Ames test-screens potential chemicals for being
  carcinogens by seeing if they are mutagens
• Bacteria are the test subjects in the Ames test.