Chapter 7

Essentials of Molecular Biology
Chapter 7
I. Genes and Gene Expression

7.1 Macromolecules and Genetic Information

 Functional unit of genetic information is the gene
 Genes in cells, composed of DNA
 Three informational macromolecules in cell
 DNA
 RNA
 Protein
 Genetic information flow can be divided into three stages
 Replication: DNA is duplicated
 Transcription: information from DNA is transferred to RNA
 mRNA (messenger RNA): encodes polypeptides
 tRNA (transfer RNA): plays role in protein synthesis
 rRNA (ribosomal RNA): plays role in protein synthesis
 Translation: information in RNA is used to build polypeptides
 Central dogma of molecular biology
 DNA to RNA to protein
 Eukaryotes: each gene is transcribed individually
 Prokaryotes: multiple genes may be transcribed together


II. DNA Structure


7.2 The Double Helix
 Four nucelotides found in DNA:
 Adenine (A)
 Guanine (G)
 Cytosine (C)
 Thymine (T)
 Backbone of DNA chain is alternating phosphates and the pentose sugar deoxyribose
 Phosphates connect 3′- carbon of one sugar to 5 of the adjacent sugar
 All cells and some viruses have DNA in double- stranded molecule
 Two strands are antiparallel
 Two strands have complementary base sequences
 Adenine always pairs with Thymine
 Guanine always pairs with Cytosine
 Two strands form a double helix

 Size of DNA molecule is expressed in base pairs
 1,000 base pairs = 1 kilobase pairs = 1 kbp
 1,000,000 base pairs = 1 megabase pairs = 1Mbp
 E. coli genome = 4.64 Mbp
 Each base pair takes up 0.34 nm of length along the helix
 10 base pairs make up 1 turn of the helix

 Inverted Repeats
 Repeated sequence that is arranged in an inverse orientation
 Stem Loops
 Short double-helical regions caused by nearby inverted repeats
 Hydrogen bonds between DNA strands hold two strands together
 Adenine–Thymine pair has two hydrogen bonds and Guanine– Cytosine pair has three hydrogen bonds
 GC pairs are stronger than AT pairs
 High heat breaks hydrogen bonds causing denaturation (melting)
 GC-rich DNA melts at higher temperatures than AT-rich DNA

7.3 Supercoiling

 Supercoiled DNA: DNA is further twisted to save space
 Negative supercoiling: double helix is underwound
 Positive supercoiling: double helix is overwound
 Relaxed DNA: DNA has number of turns predicted by number of base pairs
 Negative supercoiling is predominantly found in nature
 DNA Gyrase: introduces supercoils into DNA

7.4 Chromosomes and Other Genetic Elements

 Genome: entire complement of genes in cell or virus
 Chromosome: main genetic element in prokaryotes
 Other genetic elements include virus genomes, plasmids, organellar genomes, and transposable elements
 Viruses contain either RNA or DNA genomes
 Can be linear or circular
 Can be single or double stranded
 Plasmids: replicate separately from chromosome
 Great majority are double stranded
 Most are circular
 Generally beneficial for the cell (i.e., antibiotic resistance)
 NOT extracellular, unlike viruses
 Chromosome is a genetic element with “housekeeping” genes
 Plasmid is a genetic element that is expendable and rarely contains genes for growth under all conditions
 Presence of essential genes is necessary for a genetic element to be called a chromosome

 Transposable Elements
 Segment of DNA that can move from one site to another site on the same or different DNA molecule
 Inserted into other DNA molecules
 Three main types
 Insertion sequences
 Transposons
 Special viruses

III. DNA Replication

7.5 Templates and Enzymes

 DNA replication is semiconservative
 Each of the two progeny double helices have one parental and one new strand
 Precursor of each nucleotide is a deoxynucleoside 5′ triphosphate (dNTP)
 Replication ALWAYS proceeds from the 5′ end to the 3′ end
 DNA polymerases catalyze the addition of dNTPs
 Five different DNA polymerases in E. coli
 DNA polymerase III is primary enzyme replicating chromosomal DNA
 DNA polymerases require a primer
 Primer made from RNA

7.6 The Replication Fork
 DNA synthesis begins at the origin of replication in prokaryotes
 Replication fork: zone of unwound DNA where replication occurs
 DNA helicase unwinds the DNA
 Extension of DNA
 Occurs continuously on the leading strand
 Discontinuously on the lagging strand
 Okazaki fragments are on lagging strand

7.7 Bidirectional Replication and the Replisome

 DNA synthesis is bidirectional in prokaryotes
 Two replication forks moving in opposite directions
 DNA Pol III adds 1,000 nucleotides per second
 Replisome complex of multiple proteins involved in replication
 DNA pulled through the replisome

7.8 Proofreading and Termination
 DNA replication is extremely accurate
 Proofreading helps to ensure high fidelity
 Mutation rates in cells are 10-8–10-11 errors per base inserted
 Polymerase can detect mismatch through incorrect hydrogen bonding
 Proofreading occurs in prokaryotes, eukaryotes and viral DNA replication systems

IV. RNA Synthesis: Transcription

7.9 Overview of Transcription

 Transcription (DNA to RNA) is carried out by RNA polymerase
 RNA polymerase uses DNA as template
 RNA precursors are ATP, GTP, CTP, and UTP
 Chain growth is 5′ to 3′ just like DNA replication
 Only one of the two strands of DNA are transcribed by RNA polymerase for any gene
 Genes are present on both strands of DNA, but at different locations
 RNA polymerase has five different subunits
 RNA polymerase recognizes DNA sites called promoters
7.9 Overview of Transcription
 Promoters: site of initiation of transcription
 Promoters are recognized by sigma factor of RNA polymerase
 Transcription stops at specific sites called transcription terminators
 Unlike DNA replication, transcription involves smaller units of DNA
 Often as small as a single gene
 Allows cell to transcribe different genes at different rates

7.10 Sigma Factors and Consensus Sequences
 Sigma factors recognize two highly conserved regions of promoter
 Two regions within promoters are highly conserved
 Pribnow box: located 10 bases before the start of transcription (-10 region)
 -35 region: located ~35 bases upstream of transcription

7.11 Termination of Transcription

 Termination of RNA synthesis is governed by a specific DNA sequence
 Intrinsic terminators: transcription is terminated without any additional factors
 Rho-dependant termination: Rho protein recognizes specific DNA sequences and causes a pause in the RNA polymerase

7.12 The Unit of Transcription
 Unit of transcription: unit of chromosome bounded by sites where transcription of DNA to RNA is initiated and terminated
 Most genes encode proteins, but some RNAs are not translated (i.e., rRNA, tRNA)
 Three types of rRNA: 16S, 23S, and 5S
 rRNA and tRNA are very stable
 tRNA cotranscribed with rRNA or other tRNA
 mRNA have short half-lives (a few minutes)
 Prokaryotes often have genes related to the same process clustered together
 These genes are transcribed all at once as a single mRNA
 An mRNA encoding a group of cotranscribed genes is called a polycistronic mRNA
 Operon: a group of related genes cotranscribed on a polycistronic mRNA
 Allows for expression of multiple genes to be coordinately regulated

V. Protein Synthesis

7.13 The Genetic Code

 Translation: the synthesis of proteins from RNA
 Genetic code: a triplet of nucleic acid bases (codon) encodes a single amino acid
 Specific codons for starting and stopping translation
 Degenerate code: multiple codons encode a single amino acid
 Anti-codon on tRNA recognizes codon
 Wobble: irregular base pairing allowed at third position of tRNA
 Stop codons: signal the termination of translation (UAA, UAG, and UGA)
 Start Codon: translation begins with AUG
 Reading frame: triplet code requires translation to begin at the correct nucleotide
 Shine-Dalgarno sequence: ensures proper reading frame
 Open Reading Frame (ORF): AUG followed by a number of codons and a stop codon in the same reading frame
 Codon bias: multiple codons for the same amino acid are not used equally
 Varies with organism
 Correlated with tRNA availability
 Cloned genes from one organism may not be translated by recipient organism because of codon bias
 Some organelles and a few cells have slight variations of the genetic code (i.e., mitochondria of animals)

7.14 Transfer RNA

 Transfer RNA: at least one tRNA per amino acid
 Bacterial cells have 60 different tRNAs
 Mammalian cells have 100–110 different tRNAs
 Specific for both a codon and its cognate amino acid
 tRNA and amino acid brought together by aminoacyl-tRNA synthetases
 ATP is required to attach amino acid to tRNA
 tRNA is cloverleaf in shape
 Anti-codon: three bases of tRNA that recognize three complementary bases on mRNA
 Fidelity of recognition process between tRNA and aminoacyl-tRNA synthetase is critical
 Incorrect amino acid could result in a faulty/non-functioning protein

7.15 Translation: The Process of Protein Synthesis

 Ribosomes: sites of protein synthesis
 Thousands of ribosomes per cell
 Composed of two subunits (30S and 50S in prokaryotes)
 S = Svedberg units
 Combination of rRNA and protein
 E. coli has 52 distinct ribosomal proteins
 Translation is broken down into three main steps:
1) Initiation: two ribosomal subunits assemble with mRNA
 Begins at an AUG start codon
2) Elongation: amino acids are brought to the ribosome and are added to the growing polypeptide
 Occurs in the A and P sites of ribosome
 Translocation: movement of the tRNA holding the polypeptide from the A to the P site
 Polysomes: a complex formed by ribosomes simultaneously translating mRNA

 Steps of Translation (cont’d)
3) Termination: occurs when ribosome reaches a stop codon
 Release factors (RF): recognize stop codon and cleave polypeptide from tRNA
 Ribosome subunits then dissociate
 Subunits free to form new initiation complex and repeat process
 Many antibiotics inhibit translation by interacting with ribosomes
 Streptomycin, chloramphenicol, tetracycline, etc.
 Many antibiotics are specific for organisms from one or two domains (i.e., chloramphenicol is specific for Bacteria)

7.16 The Incorporation of Nonstandard Amino Acids

 Universal genetic code encodes 20 amino acids
 More than 100 different amino acids have been found in proteins
 Most are made through posttranslational modification
 Others are inserted during protein synthesis

7.17 Folding and Secreting Proteins

 Most polypeptides fold spontaneously into their active form
 Some require assistance from molecular chaperones or chaperonins for folding to occur
 They only assist in the folding, are not incorporated into protein
 Can also aid in refolding partially denatured proteins
 Signal sequences: found on proteins requiring transport from cell
 15–20 residues long
 Found at the beginning of the protein molecule
 Signal the cell’s secretory system
 Prevent protein from completely folding