General Biology I

Week 14 (2015.06.02)

Michael Ye ()
Associate Professor
Division of Liberal Arts & Sciences

Week

Description

Notes

1st

Introduction (3.03 & 3.05)

2nd

Chapters 1 & 2. Study of Life & Chemistry of Life (3.10 & 3.12)

3rd

Chapters 3 & 4. Chemistry of Water and Carbon (3.17 & 3.19)

Exam #1

4th

Chapter 5. Biomolecules (3.24 & 3.26)

Exam #2

5th

Chapter 6. The Cell (3.31 & 4.02)

Exam #3

6th

Chapters 7 & 8. The Cell Membrane & Metabolism (4.07 & 4.09)

Exam #4

7th

Chapters 9. Cellular Respiration (4.14 & 4.16)

Exam #5

8th

Midterm Exam

9th

Chapter 12. The Cell Cycle (4.28 & 4.30)

10th

Chapter 13. Meiosis (05.05 & 5.07)

11th

Chapter 14. The Gene (5.12 & 5.14)

Exam #6

12th

Chapter 15. Chromosomal Basis of Inheritance (5.19 & 5.21)

Exam #7

13th

Chapter 16. Molecular Basis of Inheritance (5.26 & 5.28)

Exam #8

14th

Chapter 17. From Gene to Protein (6.02 & 6.04)

Exam #9

15th

Chapter 18. Regulation of Gene Expression (6.09 & 6.11)

Exam #10

16th

Final Exam

What is a Gene?

Gene Structure (ORF)

The generic structure of a eukaryotic mRNA, illustrating some post-transcriptional

regulatory elements that affect gene expression. Abbreviations (from 5' to 3'): UTR,
untranslated region; m7G, 7-methyl-guanosine cap; hairpin, hairpin-like secondary
structures; uORF, upstream open reading frame; IRES, internal ribosome entry site;
CPE, cytoplasmic polyadenylation element; AAUAAA, polyadenylation signal.

The Central Dogma of Molecular Biology

Replication

The two functions of DNA comprise the Central Dogma of Molecular Biology:
1.

DNA can reproduce itself (replication)

2.

DNA can copy its information into RNA (transcription)

3.

RNA can specify a sequence of amino acids in a polypeptide (translation)

Basic Principles of Transcription and Translation

RNA is the bridge between genes and the proteins for which they code
Transcription is the synthesis of RNA (messenger RNA, mRNA) under the direction of
DNA
Translation is the synthesis of a polypeptide, using information in the mRNA
Ribosomes are the sites of translation
In prokaryotes, translation of mRNA can begin before transcription has finished
In a eukaryotic cell, the nuclear envelope separates transcription from translation; also,
RNA transcripts are modified through RNA processing to yield finished mRNA
A primary transcript is the initial RNA transcript from any gene prior to processing
The central dogma:

Basic Principles of Transcription and Translation

Coupled Transcription & Translation

Uncoupled Transcription & Translation

The Genetic Code

Some Important Questions
1. How are the instructions for assembling amino acids into proteins encoded into DNA? (There are
20 amino acids, but there are only four nucleotide bases in DNA)
2. How many nucleotides correspond to an amino acid?

Mechanism
The flow of information from gene to protein is based on a codon
The gene is transcribed into complementary, non-overlapping codon of mRNA, which is then
translated into amino acids (to form a polypeptide)
During transcription, one of the two DNA strands, called the template strand, provides a template
for ordering the sequence of complementary nucleotides in an RNA transcript
The template strand is always the same strand for a given gene
During translation, the mRNA base triplets or codons, are read in the 5 to 3 direction
Each codon specifies the amino acid (one of 20) to be placed at the corresponding position along a
polypeptide

The Genetic Code

Cracking the Code

All 64 codons were deciphered by the mid-1960s

Of the 64 triplets, 61 code for amino acids; 3 triplets are stop signals to end translation
The genetic code is redundant (more than one codon may specify a particular amino acid) but not
ambiguous; no codon specifies more than one amino acid
Codons must be read in the correct reading frame (correct groupings) in order for the specified
polypeptide to be produced

The Genetic Code

Degenerate & Specific: 64 codons encode 61 amino acids & 3 stop signals
Multiple codes for an amino acid tend to have two bases in common
Codons are written in a 5' 3' sequence

Marshall Warren Nirenberg

Nobel Prize in Medicine (1968)

NIH (National Institutes of Health)

With Har Gobind Khorana and Robert W. Holley

For "breaking the genetic code" and describing how it

operates in protein synthesis

In August 1961, at the International Congress of

Biochemistry in Moscow, Nirenberg presented a paper to a
small group of scientists

Poly-URIDINE (UUU): phenylalanine

Poly-ADENOSINE (AAA): lysine
Poly-CYTOSTINE (CCC): proline
Poly-GUANOSINE (GGG): glycine

Nirenberg (1962)

Har Gobind Khorana

Translation

Direction of Transcription

RNA sequences are written 5 3 (left to right)

Translation occurs 5 3 with ribosomes reading the message 5 3

Genes are written so that transcription proceeds in a left to right direction

The sequence of a hypothetical gene and the RNA transcribed from it are shown.
By convention, the gene is said to be transcribed from the 5 end to the 3 end, but
the template strand of DNA is copied from the 3 end to the 5 end. Growth of the
ribonucleotide chain proceeds 5 3

Evolution of the Genetic Code

The genetic code is nearly universal, shared by the simplest bacteria to the most
complex animals
Genes can be transcribed and translated after being transplanted from one species to
another

Molecular Components of Transcription

Transcription is the first stage of gene expression

RNA synthesis is catalyzed by RNA polymerase, which pries the DNA strands apart and
hooks together the RNA nucleotides
The RNA is complementary to the DNA template strand
RNA synthesis follows the same base-pairing rules as DNA, except that uracil
substitutes for thymine
The DNA sequence where RNA polymerase attaches is called the promoter; in bacteria,
the sequence signaling the end of transcription is called the terminator
The stretch of DNA that is transcribed is called a transcription unit

Synthesis of an RNA Transcript

The three stages of transcription
1. Initiation
2. Elongation
3. Termination

Initiation of Transcription

Promoters signal the transcriptional start point and usually extend several dozen
nucleotide pairs upstream of the start point

Transcription factors mediate the binding of RNA polymerase and the initiation of
transcription

The completed assembly of transcription factors and RNA polymerase II bound to a

promoter is called a transcription initiation complex

A promoter called a TATA box is crucial in forming the initiation complex in eukaryotes

1 A eukaryotic promoter
Promoter

Nontemplate strand

DNA
5
3

3
5

TAT AAAA
AT AT T T T

TATA box

Transcription
factors

Template strand

Start point

2 Several transcription
factors bind to DNA

5
3

3
5
3 Transcription initiation
complex forms
RNA polymerase II
Transcription factors

5
3

3
RNA transcript

Transcription initiation complex

3
5

Elongation of the RNA Strand

As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at a time
Transcription progresses at a rate of 40 nucleotides per second in eukaryotes (E.coli DNA
polymerase III incorporates about 1000 nucleotides per second)
A gene can be transcribed simultaneously by several RNA polymerases
Nucleotides are added to the 3 end of the growing RNA molecule

Termination of Transcription

The mechanisms of termination are different in bacteria and eukaryotes

In bacteria, the polymerase stops transcription at the end of the terminator and the
mRNA can be translated without further modification
In eukaryotes, RNA polymerase II transcribes the polyadenylation signal sequence; the
RNA transcript is released 1035 nucleotides past this polyadenylation sequence
Eukaryotic cells modify RNA after transcription
Enzymes in the eukaryotic nucleus modify pre-mRNA (RNA processing) before the
genetic messages are dispatched to the cytoplasm
During RNA processing, both ends of the primary transcript are usually altered
Also, usually some interior parts of the molecule are cut out, and the other parts
spliced together

Post-transcriptional Modification of mRNA

Each end of a pre-mRNA molecule is modified in a particular way

1. The 5 end receives a modified nucleotide 5 cap
2. The 3 end gets a poly-A tail

These modifications share several functions

1. They seem to facilitate the export of mRNA
2. They protect mRNA from hydrolytic enzymes
3. They help ribosomes attach to the 5 end

Split Genes and RNA Splicing

Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides
that lie between coding regions
These noncoding regions are called intervening sequences, or introns
The other regions are called exons because they are eventually expressed, usually translated into
amino acid sequences
RNA splicing removes introns and joins exons, creating an mRNA molecule with a continuous
coding sequence
In some cases, RNA splicing is carried out by spliceosomes
Spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins
(snRNPs) that recognize the splice sites

snRNPs and Spliceosomes

The Functional and Evolutionary Importance of Introns

Alternative splicing: some genes can encode more than one kind of polypeptide,
depending on which segments are treated as exons during splicing
Consequently, the number of different proteins an organism can produce is much
greater than its number of genes
Proteins often have a modular architecture consisting of discrete regions called
domains..different exons code for the different domains in a protein

Molecular Components of Translation

A cell translates an mRNA message into protein with the help of transfer RNA (tRNA)
tRNA transfer amino acids to the growing polypeptide in a ribosome
Translation is a complex process in terms of its biochemistry and mechanics

The Structure and Function of Transfer RNA

A tRNA molecule consists of a single RNA strand (80 nucleotides); tRNAs are not identical
1. Each carries a specific amino acid on one end
2. Each has an anticodon on the other end; the anticodon base-pairs with a complementary
codon on mRNA

tRNA twists and folds into a 3-D molecule (due to hydrogen bonds) into L-shaped structure; an
unfolded tRNA molecule looks like a cloverleaf

Accurate translation requires two steps

1. Correct match between a tRNA and an amino acid, done by the enzyme aminoacyl-tRNA
synthetase
2. Correct match between the tRNA anticodon and an mRNA codon

Flexible pairing at the third base of a codon is called wobble and allows some tRNAs to bind to
more than one codon

Amino acid
attachment
site

5
Amino acid
attachment
site

5
3

Hydrogen
bonds
Hydrogen
bonds

A A G

3
Anticodon

(a) Two-dimensional structure

Anticodon
(b) Three-dimensional structure

5
Anticodon

Aminoacyl-tRNA
synthetase (enzyme)

Amino acid
P
P P

Adenosine

ATP

P i

Adenosine

P i

Aminoacyl-tRNA
synthetase

P i

tRNA
tRNA

Amino
acid
P

Adenosine

AMP

Computer model

Aminoacyl tRNA
(charged tRNA)

Ribosomes

Ribosomes facilitate joining of tRNA anticodons with mRNA codons in protein synthesis

The two ribosomal subunits (large and small) are made of proteins and ribosomal RNA
(rRNA)

A ribosome has three binding sites for tRNA

1. The P site holds the tRNA that carries the growing polypeptide chain
2. The A site holds the tRNA that carries the next amino acid to be added to the chain
3. The E site is the exit site, where discharged tRNAs leave the ribosome

Growing
polypeptide

tRNA
molecules

E P

Exit tunnel

Large
subunit
A

Small
subunit

mRNA

(a) Computer model of functioning ribosome

Growing polypeptide

P site (Peptidyl-tRNA
binding site)

Exit tunnel

Next amino
acid to be
added to
polypeptide
chain

A site (AminoacyltRNA binding site)

E site
(Exit site)
E
mRNA
binding site

Amino end

Large
subunit

Small
subunit
(b) Schematic model showing binding sites

tRNA

mRNA

3
Codons

(c) Schematic model with mRNA and tRNA

Ribosome Association and Initiation of Translation

The three stages of translation (require proteins that aid in the translation process)
1. Initiation
2. Elongation
3. Termination
Initiation
The initiation stage of translation brings together mRNA, a tRNA with the first amino
acid, and the two ribosomal subunits
First, a small ribosomal subunit binds with mRNA and a special initiator tRNA
Then the small subunit moves along the mRNA until it reaches the start codon (AUG)
Proteins called initiation factors bring in the large subunit that completes the
translation initiation complex

Initiation of Translation

Elongation of the Polypeptide Chain

Elongation: amino acids are added to the preceding amino acid at the C-terminus of
the growing chain; translation proceeds along the mRNA in a 5 to 3 direction
Addition of each amino acid involves proteins called elongation factors and occurs in
three steps: codon recognition, peptide bond formation, and translocation

Termination of Translation

Termination occurs when a stop codon in the mRNA reaches the A site of the ribosome
The A site accepts a protein called a release factor which causes the addition of a water
molecule instead of an amino acid
This reaction releases the polypeptide, and the translation assembly then comes apart

Polyribosomes
Ribosomes can translate a single mRNA simultaneously, forming a polyribosome (or
polysome), which allows a cell to make many copies of a polypeptide very quickly

Targeting Polypeptides to Specific Locations

Two populations of ribosomes in cells

1. Free ribosomes mostly synthesize proteins that function in the cytosol
2. Bound ribosomes (attached to the ER) make proteins of the endomembrane system and
proteins that are secreted from the cell
Polypeptide synthesis always begins in the cytosol and finishes in the cytosol unless the
polypeptide signals the ribosome to attach to the ER
Polypeptides destined for the ER or for secretion are marked by a signal peptide
A signal-recognition particle (SRP) binds to the signal peptide and brings the signal peptide and its
ribosome to the ER

DNA Mutations

Mutations (changes in the genetic material) of one or a few nucleotides can affect protein
structure and function
Point mutations are chemical changes in just one base pair of a gene; the change of a single
nucleotide in a DNA template strand can lead to the production of an abnormal protein; two types
of point mutations within a gene
1. Nucleotide-pair substitutions
2. One or more nucleotide-pair insertions or deletions

Nucleotide Substitutions
Nucleotide-pair substitution: replace one nucleotide pair with another pair of nucleotides
1. Silent mutations have no effect on the amino acid produced by a codon because of
redundancy in the genetic code
2. Missense mutations still code for an amino acid, but not the correct amino acid
3. Non-sense mutations change an amino acid codon into a stop codon, nearly always
leading to a non-functional protein

Wild type
DNA template strand

A
T

T
A

T 5
A 3

A 3

3 T
5 A

A
T

C
G

T
A

T
A

C
G

A
T

A
T

A
T

C
G

C
G

mRNA5
Protein

Met

Lys

Phe

Gly

Stop
Carboxyl end

Amino end

(b) Nucleotide-pair insertion or deletion

(a) Nucleotide-pair substitution

Extra A

A instead of G
3 T
5 A

A
T

C
G

T
A

T
A

C A
G T

A
T

A
T

C
G

C A
G T

A
T

T
A

T 5
A 3

3 T
5 A

A
T

C
G

Met

Lys

Phe

Gly

T
A

T
A

C
G

A
T

A
T

A
T

C
G

C
G

G
C

A
T

T T 5
A A 3

U G

A
T

T
A

T 5T
A 3A

Extra U

U instead of C
5 A

A
T

A 3

5 A

Met

Stop

Silent (no effect on amino acid sequence)

Stop

Frameshift causing immediate nonsense

(1 nucleotide-pair insertion)

T instead of C
3 T
5 A

A
T

C
G

T
A

T
A

C
G

A
T

A
T

A
T

T
A

A missing

C G
G C

A
T

T
A

T 5
A 3

3 T
5 A

C
G

A
T

T
A

T
A

C
G

A
T

A C
T G

A instead of G
5 A

Met

Lys

Ser

Phe

A 3

5 A

G
C

Lys

G G

Leu

Ala

Frameshift causing extensive missense

(1 nucleotide-pair deletion)
T T C missing

A
T

C
G

A
T

T
A

C
G

A
T

A
T

A
T

C
G

C G
G C

A
T

T
A

T 5
A 3

3 T
5 A

A C
T G

U instead of A
U
Met

Nonsense

Met

Stop

A instead of T

5 A

C
G

U missing

Missense

3 T
5 A

Stop

A
T

A
T

A
T

C
G

T 5
A 3

C
G

G
C

A
T

T
A

A A 3U

A A G missing
G

A 3

A 3

5 A

U
Met

U
Phe

Gly

No frameshift, but one amino acid missing

(3 nucleotide-pair deletion)

Stop

Insertions and Deletions

Insertions (additions) and deletions (losses) of nucleotide pairs in a gene have a

disastrous effect on the resulting protein more often than substitutions do
Insertion or deletion of nucleotides may alter the reading frame, producing a
frameshift mutation

Wild type
DNA template strand 3 T

5 A

T 5
A 3

mRNA 5 A

A 3

Protein

Met

Lys

Phe

Gly

Stop

Amino end

Carboxyl end

(b) Nucleotide-pair insertion or deletion: frameshift causing immediate nonsense

Extra A
3 T
5 A

A
T

A
T

A
T

G
G

G
C

A
T

T 5
A 3

A 3

Extra U
5 A

Met

Stop

1 nucleotide-pair insertion

Wild type
DNA template strand 3 T

5 A

T 5
A 3

mRNA 5 A

A 3

Protein

Met

Lys

Gly

Phe

Stop

Carboxyl end

Amino end

(b) Nucleotide-pair insertion or deletion: frameshift causing extensive missense

A missing
3 T
5 A

T 5
A 3

U missing
5 A

Met

Lys

Leu

Ala

1 nucleotide-pair deletion

Wild type
DNA template strand 3 T

5 A

T 5
A 3

mRNA 5 A

A 3

Protein

Met

Lys

Gly

Phe

Amino end

Stop

Carboxyl end

(b) Nucleotide-pair insertion or deletion: no frameshift, but one amino acid missing
T T C missing
3 T
5 A

A
T

T
A

T 5
A 3

A 3

A A G missing
5 A

Met

Phe

3 nucleotide-pair deletion

Gly

Stop

Universality of the Gene

Gene is Universal
When comparing gene expression in bacteria, archaea, and eukarya gene expression
differs among the domains of life but the concept of a gene is universal
Archaea are prokaryotes, but share many features of gene expression with eukaryotes
Bacteria and eukarya differ in their RNA polymerases, termination of transcription, and
ribosomes; archaea tend to resemble eukarya in these respects
Transcription and Translation
Bacteria can simultaneously transcribe and translate the same gene
In eukarya, transcription and translation are separated by the nuclear envelope
In archaea, transcription and translation are likely coupled

Coupled Transcription and Translation

Sample Problems

In-Class Problem #1
C
T
C

DNA Double Helix

mRNA Codon
G

Tryptophan

tRNA Anticodon
Amino Acid

In-Class Problem #2
A double-stranded DNA molecule with the sequence shown here produces, in
vivo, a polypeptide that is five amino acids long.
TAC ATG ATC ATT TCA CGG AAT TTC TAG CAT GTA
ATG TAC TAG TAA AGT GCC TTA AAG ATC GTA CAT
a. Which strand of DNA is transcribed and in which direction?
b. Label the 5 and the 3 ends of each strand.