The Blueprint of Life, From DNA to Protein

1
The Blueprint of Life, From DNA to Protein

• Chapter 7

2
7.1 Overview

• Genome - complete set of genetic information of a
  species
• Gene - the functional unit of the genome
• Transmission of heritable information
• Encodes polypeptides or functional RNA

3
Characteristics of DNA

• The central dogma of molecular biology

4
Characteristics of DNA

• Deoxyribose-phosphate backbone
• Nitrogenous bases
• Double-stranded (complementary)
• Hydrogen bonds between bases of strands
• Base pairing
• AT (two H bonds)
• GC (three H bonds)
• Polarity (5 to 3)
• Strands are anti-parallel

5
Characteristics of DNA
6
Characteristics of RNA

• Ribose-phosphate backbone
• Usually single stranded
• Can form intrachain base pairs
• Can fold into structures with catalytic activity
• U replaces T
• RNA is transcribed from DNA
• A complementary copy
• Termed transcript

7
Characteristics of RNA

• RNA Transcripts
• Messenger RNA (mRNA) encodes proteins
• mRNA is read in groups of 3 nucleotides, termed a
  codon, which determines amino acid composition of
  a polypeptide
• Ribosomal RNA (rRNA) fold into 3D structures with
  catalytic activities
• Transfer RNA (tRNA) binds to and specifies amino
  acids for translation

8
Regulating Gene Expression

• Cells have the ability to regulate the magnitude
  of mRNA synthesis
• This regulation is mediated by a large group of
  proteins that interact with the DNA to fine-tune
  expression of genes
• These proteins act as a dimmer switch more than
  an on/off switch
• Their activities are largely controlled by
  environmental influences

9
7.2 DNA Replication

• Bacteria have circular chromosomes of DNA
• Prior to cell division, the chromosome must be
  replicated (aka - duplicated)
• This process is bidirectional due to the
  anti-parallel nature of double-stranded DNA
• The replication is semiconservative because each
  strand acts as a template for the synthesis
• After replication, each chromosome is composed of
  one template (parental) strand and one new strand

10
DNA Replication

• Steps
• DNA strands separate (denature) and unwind
  because of the activities of helicases
• RNA primer hybridizes to the template DNA at
  origin of replication sites
• Primers are short single-stranded
  oligonucleotides no more than a few dozen bases
  in length that are synthesized by the enzyme
  primase
• Hybridization occurs by complementary base pairing

11

• Steps (cont.)
• DNA polymerases bind to the primer-template and
  inserts a new base that is complementary to the
  template base
• The new bases are deoxynucleotide triphosphates
  (dNTP) and their incorporation results in the
  loss of the ß and ? phosphates
• The a phosphate becomes the phosphate backbone

dATP dTTP dGTP dCTP
12
DNA Replication

• Steps (cont.)
• The template strand is read 3 to 5
• The new strand is synthesized 5 to 3
• To accommodate winding tension, DNA gyrases relax
  the coiled DNA by breaking it ahead of the DNA
  polymerase
• This process of replication results in a
  replication fork where the parental strands are
  separating and the new strands are being
  synthesized

13
(No Transcript)
14
DNA Replication

• Okazaki fragments
• Although one strand can be synthesized
  continuously, the other strand cannot
• These strands are referred to as the leading
  strand and lagging strand, respectively
• The lagging strand is synthesized in fragments of
  several hundred nucleotides in length
• These fragments are termed Okazaki fragments

15
DNA Replication
DNA Synthesis
16
DNA Replication
Nucleotide Incorporation
17
7.3 Gene Expression

• Transcription
• The plus strand encodes the genetic information
• The minus strand is read by the RNA polymerase to
  make RNA
• This is because nucleic acids are complementary

18
Transcription

• Upstream (i.e, 5) of all genes is the promoter
  region
• The promoter contains sequences of DNA that are
  recognized by regulatory proteins, termed
  transcription factors
• These are the dimmer switch sites for controlling
  the magnitude of gene expression

19
Transcription

• In prokaryotes, an mRNA molecule can encode more
  than one polypeptide
• monocistronic - encodes one polypeptide
• polycistronic - encodes two or more polypeptides

20
Transcription

• Initiation
• The sigma factor is a subunit of RNA polymerase
  that recognizes promoters
• After RNA pol binds to the promoter, sigma
  dissociates, leaving the RNA pol core enzyme
• Elongation
• RNA pol reads the minus strand of the gene and
  synthesizes the mRNA 5 to 3
• At the 3 end of the gene is a termination
  sequence that displaces the RNA polymerase from
  the gene, thus ending transcription

21
Transcription
22
Translation

• Translation is the process of reading the mRNA
  and synthesizing a polypeptide
• Each mRNA has a short 5 untranslated region
  (UTR), a coding region, and a long 3 UTR
• The coding region is read three nucleotides at a
  time (codons) to determine the amino acids and
  their order
• The UTRs contain regulatory elements that control
  several aspects of mRNA activities, including its
  half-life

23
Translation

• The Genetic Code
• 20 amino acids must be encoded by a 4-letter
  alphabet (A, U, G, C)
• The minimum word size for 20 amino acids is 3
  letters (i.e., 4216 4364)
• AUG is the universal start codon
• There are 3 stop codons
• The code is degenerate because more than one
  codon can encode some amino acids

24
Translation

• Ribosomes are the sites of polypeptide synthesis
• Ribosomes are composed of RNAs and proteins
• They assemble from two large complexes with the
  mRNA to form a single complex
• The Shine-Delgarno sequence at 5 UTR assures
  proper alignment of the mRNA on the ribosome
• This allows the ribosome to find the AUG start
  codon

25
Translation

• Transfer RNA molecules contain two important
  features
• An anti-codon that is complementary to the codon
• An amino acid that is specific for the codon

26
Translation

• The anticodon of the tRNAiMet aligns with start
  codon
• The second tRNA aligns with the second codon
• A ribozyme (catalytic RNA molecule) termed
  peptidyl transferase forms a peptide (covalent)
  bond between the COOH of the first amino acid and
  the NH2 of the second amino acid
• The first amino acid (Met) is released from its
  tRNA and the tRNA is ejected from the ribosome
• The ribosome moves to the next codon and repeats
  the elongation
• When a stop codon is encountered, translation is
  terminated and the polypeptide is released from
  the ribosome

27
Translation
28
7.4 Differences Between Eukaryotic and
Prokaryotic Gene Expression
29
7.5 Prokaryotic Gene Regulation

• Some genes are constitutively expressed
• Most are modulated
• Repressors bind to the DNA between the promoter
  and transcriptional start site, thus block RNA
  pol
• Activators interact with the promoter region to
  facilitate RNA pol binding

30
The lac Operon

• The lac operon is a sensing pathway for lactose
• The presence of lactose and absence of glucose in
  culture media stimulates the expression of genes
  that metabolize lactose
• As glucose levels drop, cyclic AMP increases
• cAMP binds to catabolite activating protein
  (CAP), allowing it to bind to the promoter
• Allolactose, an isomer of lactose, binds to the
  lac repressor, preventing from binding to the DNA
• RNA pol binds to the DNA and begins mRNA synthesis

31
The lac Operon
32
7.6 Sensing and Responding to Environmental
Fluctuations

• Microbes must adjust to environmental changes
• This typically occurs because of the presence of
  specific macromolecular receptors on the surface
• Ligation to these receptors induces a series of
  signal transduction events inside the cell that
  results in a change in gene expression profile
• The expressed genes play a role in managing the
  environmental changes

33
7.7 Genomics

• A principal focus of genomics is to sequence
  entire genomes of organisms
• Several microbial genomes have been sequenced and
  has permitted an analysis of important genes
• The sequencing of medically-important genomes
  will permit better understanding of pathogenesis,
  antibiotic susceptibility and vaccine development

34
(No Transcript)