Chapter 6

Genes

• Functional unit of genetic information

• Genes are a part of genetic elements: large molecules and or chromosomes

Nucleic acids

• contain genetic information via nucleotides (monomers of nucleic acids)

Nucleoside

has pentose sugar and nitrogen base, no phosphate

Nucleotide

nucleic acid monomers, DNA and RNA are polynucleotides, Three components- pentose sugar (ribose or deoxyribose), nitrogenous base, phosphate (PO43-)

Four nucleotides found in DNA

Nitrogenous bases are pyrimidines or purines

• adenine (A) (a purine in DNA and RNA)

  – guanine (G) (a purine in DNA and RNA)

  – cytosine (C) (a pyrimidine in DNA and RNA)

  – thymine (T) (a pyrimidine in DNA)

  – AGC also found in RNA but not T.

  – uridine (U) (pyrimidine found in RNA)

Properties of the Double Helix

• DNA is double stranded and help by hydrogen bonding between bases

  • Complementary base sequences

    • A and T

    • G and C

  • Two strands are antiparallel (5’-3’ and 3’-5’)

    • Contains two grooves, major (where protein binds) and minor

DNA and RNA

• genetic blue print

• transcription product

Properties of RNA

• Contains ribose

• Single stranded

• Base pairings

  • A and U

  • G and C

Topoisomerases

insert and remove supercoils

Negative supercoiling

twisted in opposite sense relative to right-handed double helix

DNA gyrase

Introduces supercoils into DNA via double-strand breaks

Positive supercoiling

Helps prevent DNA melting at high temps

Central Dogma

theory stating genetic information flow can be divided into three stages, DNA to RNA to Protein

Gene expression

Transfers DNA information to RNA

Three main RNA classes involved in protein synthesis

mRNA (messenger)

• carry information to ribosome

tRNA (transfer)

• convert mRNA information to amino acid sequence

rRNA (ribosomal RNA)

• catalytic and structural ribosome components

• help hold ribosomal proteins in place and help locate the beginning and end of the mRNA message

Three stages of information flow

Replication

Transcription

Translation

Replication

DNA is duplicated by DNA polymerase

Transcription

Information from DNA is transferred to RNA by RNA polymerase

Translation

Information in mRNA is used to build polypeptides on ribosome

Eukaryotes

• Each gene is transcribed individually into a single mRNA

▪ Replication and transcription occur in nucleus

▪ RNAs must be exported outside nucleus for translation

Prokaryotes

▪ Multiple genes may be transcribed in one mRNA

▪ Coupled transcription and translation occur producing proteins at maximal rate

– Some viruses violate central dogma

Why is DNA replication considered semi-conservative?

- Each of the two resulting double helices has one new strand and one parental (template) strand

Why are DNA strands considered anti-parallel?

- The two strands go in opposite directions. Needed to form the hydrogen bonds between the nitrogenous bases.

Chromosome

Main genetic element in prokaryotes

Most bacteria and archaea have single circular chromosome carrying all/most genes

Viruses

Contain either RNA or DNA genomes

• can be single or double stranded

• can be linear or circular

Plasmids

• circular or linear double-stranded DNA that replicate separately from chromosome

• usually circular, extrachromosomal

• NOT extracellular, unlike viruses

• generally beneficial for the cell

• found in many bacteria and archaea

• mostly nonessential for cell function under all conditions

Different types of plasmids

a. R plasmids are a well-studied type of plasmid that confers resistance to antibiotics or other growth inhibitors.

b. In pathogenic bacteria, plasmids can encode virulence factors, bacteriocins, and play a role in metabolism. Bacteriocins are proteins that inhibit or kill closely related species of different strains of the same species

c. Plasmids are important for conjugation, a mechanism of horizontal gene transfer.

Transposable elements

• segments of DNA inserted into other DNA molecules that can move from one site to another site on the same or a different DNA molecule (e.g., chromosomes, plasmids, viral genomes)

• inserted into other DNA molecules (e.g., chromosomes, plasmids, viral genomes)

• found in prokaryotes and eukaryotes

Virulence factors

• ability to attach or produce toxins

Bacteriocins

• proteins that inhibit or kill closely related species or different strains of the same species

Precursor of each nucleotide is

deoxynucleoside 5’-triphosphate (dNTP)

Replication always proceeds from the

5’ end to the 3’ end

DNA polymerases

• Catalyze polymerization of deoxynucleotides

• Can only add nucleotides to preexisting 3’-OH and require a primer: short stretch of RNA

  • Primer made for RNA by DNA primase

  • Primer eventually removed and replaced with DNA

DNA polymerase III

primary enzyme for DNA replication

DNA ligase

seals nicks in DNA

DNA helicase

Unwinds double helix at replication fork

DNA primase

Primes new strands of DNA

RNA primer

a nucleic acid molecule to which DNA polymerase can attach the first nucleotide

Initiation of DNA Synthesis

• Double helix must be unwound to expose template strands at the replication fork

• DNA helicase unwinds double helix

• DNA synthesis begins where DNA protein binds and opens double helix

• Helicase (DnaB) and loader protein (DnaC) bind

• Primase and DNA polymerase enzymes loaded and DNA replication begins

• Replication fork moves along DNA

Leading and lagging strands and the replication process

Leading Strand:

• Replication occurs continuously from 5' to 3'.

• Always has a free 3'-OH end.

• No need for RNA primers.

Lagging Strand:

• Replication is discontinuous

• No free 3'-OH end.

• Primase synthesizes multiple RNA primers on this strand.

• Primase replaced by DNA Pol III, DNA synthesis continues until it reaches previously synthesized DNA

• DNA polymerase I removes the RNA primer and replaces it with DNA

• DNA ligase seals nicks in the DNA

DNA synthesis is bidirectional in prokaryotes

because of circular chromosome

• two replication forks moving in opposite directions

• DNA Pol III adds 1000 nucleotides per second

Fidelity of DNA replication

DNA replication is highly accurate

Errors can lead to mutations, causing change in DNA sequence

Proofreading

helps to ensure high fidelity

Proof reading process

Detection of Mismatch:

• DNA Pol I and Pol III detect base pair mismatches during replication.

Distortion Recognition:

• Recognition through double helix distortion.

Exonuclease Activity:

• 3' to 5' exonuclease removes mismatched bases.

Reinsertion:

• DNA Pol I and Pol III insert the correct bases.

Detection of mismatch

DNA Pol I and Pol III detect base pairs mismatches during replication

Distortion Recognition

Recognition through double helix distortion

Exonuclease activity

3’ to 5’ exonuclease removes mismatched bases

Reinsertion

DNA pol I and pol III insert the correct bases

Exonuclease Proofreading

Occurs in prokaryotes, eukaryotes and viral DNA replication systems

Transcription

• Process of synthesizing RNA from a DNA template

  • RNA polymerase carries out transcription

  • transcription makes different types of RNA

    • mRNA, tRNA, rRNA, and regulatory RNAs

RNA

Precursors are ATP, GTP, CTP, and UTP

Promoter sequence

-Initiation sites on DNA that initiate the transcription of a particular gene

Consensus sequence

• a representation of the most common nucleotide or amino acid at each position in a sequence alignment

• For the Pribnow box (-10 region) is typically TATAAT

• sequence is recognized by RNA polymerase during transcription initiation, signaling the start site for transcription.

Sigma Factor of RNA polymerase

recognizes initiation sites on DNA

Start codon sequence

Translation begins with AUG

Encodes N-formyl methionine in Bacteria and methionine in Archaea and Eukarya

AUG codes for methionine in Archaea and Eukarya

Where does RNA polymerase bind DNA?

DNA sequence known as the promoter region

Transcriptional units

DNA segments transcribed into 1 RNA molecule bounded by initiation and termination sites

Polycistronic mRNA

Operons transcribed into a single mRNA, contain multiple open reading frames that encode amino acids

Factor dependent termination

• Rho protein recognizes specific DNA sequences (Rho-dependent termination site) and releases RNA polymerase from DNA

Factor independent termination

A specific sequence in the mRNA, known as the terminator sequence, is responsible for termination without the need for additional proteins.

Rho factor

Protein factor involved in the termination of transcription in bacteria

Polysome

a single mRNA molecule

• Increases both speed and efficiency of translation because each ribosome in the polysome makes a complete polypeptide

16s rRNA

• rRNA facilitates initiation via base pairing, holds mRNA in position on either side of A and P sites. Also involved in ribosome subunit association

23s rRNA

considered a ribozyme because it possesses catalytic activity, and it plays a direct role in the peptidyl transferase reaction during protein synthesis. Catalyzes peptidyl transferase reaction

Transposable elements

• Segments of DNA inserted into other DNA molecules that can move from one site to another site on the same or a different DNA molecule (e.g., chromosomes, plasmids, viral genomes)

• Inserted into other DNA molecules (e.g, chromosomes, plasmids, viral genomes)

• Found in prokaryotes and eukaryotes

Operon

two or more genes transcribed under control of promoter region (single regulatory site) located upstream of where RNA polymerase initiates transcription

Regulon

Multiple operons that encode genes whose products are needed under the same conditions. Respond to a specific signal by a single regulatory protein by turning on or off

Stem-loop structure

• An intramolecular base pairing that can occur in single stranded DNA or RNA if sequences of two regions of the same strand are complementary to each other. two stretches of nucleotides near each other are complementary and can thus base-pair

Inverted repeat

• a nucleotide sequence followed downstream by its inverted complement

Proteins

• catalytic proteins (e.g., enzymes)

  – structural proteins (e.g., parts of membranes, cell

  envelope, ribosomes)

  – regulatory proteins (e.g., DNA binding, affecting

  transcription)

  • Proteins are polymers of amino acids.

  • Amino acids are linked by peptide bonds to form a polypeptide.

  • Proteins are one or more polypeptide(s).

Primary structure

Linear array of amino acids in a polypeptide

Secondary structure

Formed from hydrogen bonding (alpha-helix or beta sheet)

Tertiary structure

three-dimensional shape of polypeptide from hydrophobic and other interactions

Quaternary structure

number and types of polypeptides (subunits) that make a protein

Denaturation

loss of structure and biological properties

Transfer RNA (tRNA)

• carry amino acids to the ribosome during translation.

• Cloverleaf structure

• The anti-codon region of tRNA recognizes and base pairs with the cognate codon in the mRNA during the translation process

Anticodon

Three bases that recognize codon (three nucleic acids encoding an amino acid)

Recognition of tRNA by

Aminoacyl-tRNA synthetase is critical for translation fidelity

• requires specific contact

• incorrect amino acid could result in a faulty or no functioning protein

Genetic code

A triplet of nucleic acid bases (codon) encodes for a specific amino acid

• 64 possible codons

• specific codons for starting and stopping translation

Degenerate code

Multiple codons encode a single amino acid

• some tRNAs recognize more than one codon

• Wobble

  • irregular base paring allowed at third position of tRNA

Codon bias

Multiple codons for the same amino acid are not used equally

• varies between organisms

• correlated with tRNA concentration

Reading frame

Triplet code requires translation to begin at the correct nucleotide

Shine-Dalgarno sequence/Ribosome-binding site

ensures proper reading frame in bacteria

Stop (nonsense) codons

Terminate translation (UAA, UAG,UGA)

Open reading frame

AUG followed by a number of codons and a stop codon

Ribosomes

Sites of protein synthesis

• thousands of ribosomes per cell

The mechanism of protein synthesis

initiation, elongation, termination

• Uses mRNA, tRNA, ribosomes

• requires multiple proteins

• Needs GTP for energy (guanosine triphosphate)

Initiation of Translation

• Begins with fee 30S ribosomal subunit

• Initiation complex forms

  • includes 30s, mRNA, formyl methionine, tRNA, and initiation factors

Ribosomal binding site

• located 3-9 nucleotides towards the 5’ end of mRNA

• Complementary to sequence on the 3’ end of 16s rRNA

Initiation Steps

• Two ribosomal subunits + Formyl methionine tRNA+ Initiation factors assemble the mRNA

• Initiation begins at an AUG start codon

Elongation

• Amino acids are brought to the ribosome and added to the growing polypeptide

• Occurs in the A(acceptor) and P(peptide) sites of ribosome

• Growing polypeptide moves to tRNA at the A site as a new peptide bond is formed

Termination

• Occurs when the ribosome reaches a stop codon

• Release Factors

  • recognize stop codon and cleave the polypetide from tRNA

• Ribosome subunits then dissociate

• Subunits are free to form a new initiation complex and repeat the process

Chaperone

a protein that assists in the folding or unfolding of other proteins, preventing misfolding and aggregation

Function of Chaperones

o Catalyze macromolecular folding events

o Refold partially denatured proteins

o Heat shock proteins,

• Synthesized at an accelerated rate when cells are stressed by heat.

• Heat shock response is an attempt to refold partially denatured proteins caused by elevated temperature

o Cold shock proteins

• produced at very cold temps, function as RNA chaperones, facilitating RNA folding and stability

o Assembling cofactor containing enzymes

The Sec System

• Exports unfolded proteins

• Inserts integral membrane proteins into the cytoplasmic membrane

The Tac System

• Transports previously folded proteins through the cytoplasmic membrane

Gram-negative systems

Types I, III, IV, VI

• One step translocases

  • move proteins in single step

• Form channels through both cytoplasmic and outer membranes

• Do not require Sec or Tat

Type I

includes cytoplasmic membrane transporter, outer membrane pore, and membrane fusion protein; requires A TP

Type III

Injects toxins into eukaryotic host cells

Type IV

most common and normally transfers DNA through conjugation; pilus-like