Nucleotides, Nucleic Acids, and Protein Synthesis Overview

Gene

segment of DNA that contains the information required for the synthesis of functional biological products, whether protein or RNA.

rRNA

carry out protein synthesis of proteins.

mRNA

intermediaries; carry information for the synthesis of proteins from genes to ribosomes.

tRNA

adapter molecules that translate information in the mRNA into a specific sequence of amino acids.

ncRNA

noncoding RNA.

Nucleotide

consists of a nitrogenous base (purine or pyrimidine), a pentose sugar, and one or more phosphate groups.

Nucleoside

combination of the pentose sugar and the nitrogenous base when no phosphate is present.

N-β-glycosyl bond

bond formed between the base joined covalently at N-1 of pyrimidines and N-9 of purines to the 1' carbon of the pentose.

Phosphodiester linkage

joins nucleotides together between the 5'-hydroxyl group of one pentose and the 3'-hydroxyl group of the next.

RNA

nucleic acid that contains ribose, with common pyrimidine bases uracil and cytosine.

DNA

nucleic acid that contains 2'-deoxyribose, with common pyrimidine bases thymine and cytosine.

Purines

adenine and guanine.

Pyrimidines

cytosine, thymine, and uracil.

Deoxyribonucleotides

structural units of DNA: A, dA, dAMP - deoxyadenosine; G, dG, dGMP - deoxyguanosine; T, dT, dTMP - deoxythymidine; C, dC, dCMP - deoxycytidine.

Ribonucleotides

structural units of RNA: A, AMP - Adenosine; G, GMP - Guanosine; U, UMP - Uridine; C, CMP - Cytidine.

Tautomers

isomers that interconvert rapidly such that they exist in equilibrium.

Hydrophobic bases

bases that are insoluble in water at neutral pH and stack to minimize contact with water.

Modified bases

found in DNA and RNA, regulating or protecting genetic information.

Exocyclic

not within the ring structure.

Phosphate-group bridges

covalently bind nucleotides where the 5' phosphate group joins to the 3'-hydroxyl group of the next nucleotide.

Oligonucleotide

short nucleic acid (50 or fewer nucleotides).

Polynucleotide

longer nucleic acids.

Avery-MacLeod-McCarty experiment

showed that DNA isolated from one bacterial strain can enter and transform the cells of another strain.

Hershey-Chase experiment

Showed that the DNA of the bacterial virus, but not its protein coat, carries the genetic message for replication of the virus in a host cell.

Primary structure of nucleotides

Covalent structure and nucleotide sequence.

Secondary structure of nucleotides

Regular, stable structure taken up by some or all of the nucleotides in a nucleic acid.

Tertiary structure of nucleotides

Complex folding of large chromosomes within eukaryotic chromatin and bacterial nucleoids; the elaborate folding of large tRNA and rRNA molecules.

Chargaff's Rules

The number of one residue will equal the number of matching residues (example: # of A = # of T and the # of G = # of C), which means that A + G = T + C.

Watson and Crick model

Postulated that native DNA consists of two antiparallel chains in a right-handed double-helical arrangement.

Complementary base pairs

A=T and G=C, formed by hydrogen bonding between chains in the helix.

Base pair stacking

Base pairs are stacked perpendicular to the long axis of the double helix, 3.4 Å apart, with 10.5 base pairs per turn.

Deoxyribose and phosphate groups

Hydrophobic, facing the water on the outside of the DNA structure.

C-2' endo conformation

The furanose ring of each deoxyribose is in this conformation.

G-C bonds

3 bonds can form between G and C, making it stronger than the 2 bonds that can form between A and T.

Stability of DNA

DNA has a higher content of G and C because it's more stable.

B-DNA

The standard form of DNA, most stable structure.

A-DNA

Favored in solutions that don't contain water; wider helix with 11 base pairs rather than 10.5.

Z-DNA

Left-handed helical rotation, 12 base pairs per helical turn, with a zigzag backbone.

Purine conformations

Purine has 2 stable conformations called syn (both groups on the same side) and anti (groups on opposite sides).

Pyrimidine conformation

Pyrimidine is stuck in anti due to steric hindrance between the sugar and the carbonyl oxygen.

Hairpin structure

Complementary parts of a palindromic repeat fold back and pair to form an antiparallel duplex helix closed at one end.

Cruciform structure

Each separated strand is paired internally to form opposing hairpin structures.

Mirror repeats

Inverted repeat occurs within each individual strand of DNA; cannot form hairpin or cruciform structures.

mRNA

Transfers genetic information to DNA to ribosomes for protein synthesis.

Transcription

mRNA is formed on a DNA template by using the genetic information contained on one strand of DNA to specify a complementary sequence of bases in an mRNA.

Ribosome function

Once mRNA reaches the ribosome, it gives the template to the amino acid in the polypeptide chain.

Monocistronic

carries the code for one polypeptide (most eukaryotic cells are this)

Polycistronic

carries the code for 2 or more polypeptides

Amino acid coding

Each amino acid is coded by at least 3 nucleotides therefore it's a 1:3 ratio

tRNA

pairs with mRNA to grow polypeptide in the correct sequence.

rRNA

are component of ribosomes.

Noncoding RNA (ribozymes)

have enzymatic activity.

RNA structure

RNA is structurally complex; single RNA strands can fold into hairpins, double-stranded regions, or complex loops.

Base pairing in RNA

Base pairing between G and U is allowed when two single strands of RNA pair with each other.

A-form helix

When perfectly complementary, the helix is an A-form (uncommon).

Z-form helix

Z-form has been made in a lab.

B-form helix

B-form has not been observed.

Melting of DNA

Native DNA undergoes reversible unwinding and separation of strands (melting) upon heating or at extreme pH.

Melting point and base pairs

DNAs rich in pairs have higher melting points than DNAs rich in pairs.

Hyperchromic effect

increase in absorption.

RNA stability

RNA is more stable though so it takes more heat to denature.

DNA mutations

permanent changes in genetic material.

AP site

Base is lost, creating a DNA lesion called an AP site or abasic site.

Deaminating agents

cause damage.

Alkylating agents

methylate guanine to make methylguanine, which can't base pair.

Oxidative damage

is the most common alteration made.

Oligonucleotides

of known sequence can be synthesized rapidly and accurately.

Polymerase chain reaction (PCR)

provides convenient and rapid method for amplifying segments of DNA if the sequences of the ends of the targeted DNA segment are known.

Requirements for PCR

Requires DNA sample, synthetic oligonucleotide primer, pool of deoxynucleoside triphosphates, and a DNA polymerase.

Sanger dideoxy sequencing

Routine DNA sequencing of genes or short DNA segments is carried out using an automated variation of Sanger dideoxy sequencing.

Phosphite linkage

A bond formed during the reaction with immobilized nucleotide, leading to the elimination of the diisopropylamino group.

Next-gen sequencing technologies

Commercial methods that allow for the efficient determination of DNA sequences, including entire genomes, in hours or days.

Sanger sequencing

A method of DNA sequencing that uses dideoxy chain termination to stop DNA strand extension.

dNTPs

Normal nucleotides used in DNA polymerase reactions.

ddNTPs

Modified nucleotides that stop DNA strand extension and are fluorescently labeled for identification.

ATP

The central carrier of chemical energy in cells.

Cyclic AMP

A common second messenger formed from ATP in a reaction catalyzed by adenylyl cyclase.

DNA cloning

The process of generating a DNA fragment of interest, inserting it into a cloning vector, and transferring it into a host cell for replication.

Recombinant DNA

Composite DNA formed by the assembly of DNA segments in new combinations.

Restriction endonucleases

Enzymes that cleave DNA into smaller fragments at specific sequences.

DNA ligase

An enzyme that links the cloning vector to the DNA fragments to be cloned.

Reverse transcriptase

An enzyme that makes a DNA copy of an RNA molecule.

Polynucleotide kinase

An enzyme that adds a phosphate to the 5'-OH end of DNA to label it or permit ligation.

Terminal transferase

An enzyme that adds homopolymer tails to the 3'-OH end of DNA.

Exonuclease III

An enzyme that removes nucleotide residues from the 3' ends of DNA.

Sticky ends

Staggered cuts on the two DNA strands, leaving 4 unpaired strands.

Blunt ends

Cuts that cleave the DNA straight across, leaving no unpaired bases on the ends.

Cloning vectors

DNA molecules used to transport foreign genetic material into another cell.

Plasmids

Circular DNA that replicates separately from the host chromosome.

BACs

Bacterial artificial chromosomes that can clone large segments of DNA.

YACs

Yeast artificial chromosomes used to clone very long segments of DNA.

Shuttle vectors

Plasmids that can propagate in cells of two or more species.

Expression vectors

Vectors that incorporate cloned genes with the sequence signals needed for transcription and translation.

Expression systems

Systems used to express proteins in different types of cells, including bacteria, yeast, insects, and mammalian cells.

Genetic engineering techniques

Methods that can alter cloned genes as required by the investigator.

Fusion protein

Proteins or peptides can be attached to a protein of interest by altering its cloned gene.

Affinity chromatography

Methods used to purify proteins by utilizing additional peptide segments.

Polymerase chain reaction (PCR)

Permits the amplification of chosen segments of DNA or RNA for cloning.

RT-PCR

Can be used to derive sequences from living cells instead of dead ones.

qPCR

Estimates the relative copy numbers of particular sequences in samples.