biochem exam 3

what is tRNA?

transfer RNA and is the carrier of activated amino acids to the ribosome for protein synthesis

what is rRNA?

ribosomal RNA and is the structural and catalytic component of ribosomes. most abundant of the RNAs

what is the role of other RNA molecules?

to regulate gene expression and process RNA molecules

how does RNA polymerase synthesize RNA molecules?

in the 5’ to 3’ direction which is similar to that of DNA polymerases but requires NTPs. they also do not need primers which provides limited proofreading activity.

what is a nucleotide?

they are energy carriers, enzyme cofactors in oxidation-reduction reactions, and intracellular signaling molecules

what are nucleic acids?

storage and transfer of genetic information, DNA & RNA, structural and catalytic roles (ribozymes), and regulation of gene expression

what is the chemical structure of a nucleotide?

sugar, base, and one or more phosphate groups

where is the base attached to the sugar?

C-1’ through an N-glycosidic linkage in the beta configuration

where are the phosphoryl groups attached?

C-5’ of nucleotide sugar

what makes ribose different from deoxyribose?

lacks the 2’ OH group

what is the difference between a purine and pyrimidine?

purine —> 2 rings fused, pyrimidines —> single 6-membered ring

what is a nucleoside?

base and sugar

what is the structure of ATP?

it is a nucleoside triphosphate and is synthesized from ADP through oxidation of metabolic fuels or by photosynthesis (recycled 50-75kg in humans)

how are nucleic acids related to nucleotides?

they are polar polymers joined by 3’ to 5’ phosphodiester linkages

what is the pka of the phosphate group and how does that relate to the backbone?

2.0 and highly negatively charged

which of the ends are phosphorylated?

5’

How is RNA susceptible to hydrolysis?

under basic conditions through an intramolecular reaction that involves the 2’ OH group

what does RNA hydrolysis form?

a cyclic monophosphate intermediate

what is the watson-crick model of DNA?

DNA consists of two polynucleotide strands that wind around each other to form a right-handed double helix

where are the sugar-phosphate backbones found in DNA?

exterior of helix and exposed to solvent

how many base pairs are in a turn of helix?

10.4

what is the helical pitch of DNA?

34 angstroms

how many rises per base pair?

3.4 angstroms

what is the diameter of the helix (helical width)

20 angstroms

what forces stabilize the DNA double helix?

H bonds with base pairs, stacking interactions (hydrophobic

which are the strongest stacking interactions?

GC base pairs

what is Tm?

denaturation of DNA occurs at high temperatures or by changing the ionization state of its bases. anything that weakens or denatures will cause a decrease in Tm

what are the structural differences of A-, B-, Z- DNA?

A&B are right-handed, Z is left-handed. B is the Francis and Crick of DNA

what are the grooves in DNA?

major —> wider and deeper and binds to many proteins

what is the feature of RNA molecules?

single-stranded, can fold back on themselves to form stem-loops, bulges, and other types of secondary and tertiary structures

where does DNA synthesis proceed?

in 5’ to 3’ direction and requires a template strand, dNTPS, polymerase w/ magnesium ions, and a primer with a 3’ OH group

how is DNA synthesized?

by DNA polymerase catalyzing the synthesis of a new strand of DNA from dNTPs and a templat strand

what causes the release of PPi?

the 3’OH group attacking the a phosphorus of the incoming dNTP

what is the role of magnesium in DNA synthesis?

they are cofactors in the active site of DNA polymerase and facilitate the reaction

what is DNA replication?

bidirectional, semi-conservative, and semi-discontinuous

what is the role of hydrolysis PPi for polymerase?

drives the reaction forward at delta G -19kj/mol

what is the purpose of polymerase proofreading?

to ensure replication occurs with high fidelity because polymerase can make a mistake every 1/10 times.

what is mRNA?

it is messenger RNA and is the template for protein synthesis (a small fraction of total RNA in cells)

what are restriction enzymes

enzymes found in prokaryotic organisms that cleave foreign DNA sequences

how can dna fragmentation be separated?

gel electrophoresis on an agarose or polyacrylamide gel

what is southern blotting?

when DNA fragments are separated by electrophoresis and then denatured and transferred to a polymer sheet with a DNA probe

what is the dideoxy (Sanger) method?

chain termination of dideoxynucleotides which are separated by capillary electrophoresis and detected by fluorescence. need a priming strand w/ 3'OH and can sequence 1000 bases in a single run

what is the illumina/solexa?

uses nucleotides and are labeled with different dyes that can be washed and repeated

what is the 3’ blocker reversible terminatiors?

it is an repetitive process bc of DNA sequencing which allows it to be conducted in short periods of time

what are important applications of pcr?

virus and bacteria detection and DNA fingerprinting in forensics

how is recombinant DNA formed?

two DNA molecules are cut with the same restriction enzymes or different enzymes that produce compatible ends

what joins the ends for recombinant DNA molecules

DNA ligase

how is cloning for recombinant DNA formed in bacteria

plasmid is cut with restriction enzymes and gene of interest is entered which is put back into bacteria via transformation

what is site-directed mutagenesis?

mutagenic primers are uses to generate a mutant plasmid by pcr, uses a mismatched base and is transformed into bacteria

what is deletion mutagenesis

pcr primers that flank the sequences to be deleted are used

what is real-time pcr

rna is isolated and reverse transcribed to produce cdna which is tgeb amplified with primers specific to the sequence of interest in the presence of a DNA-binding dye. helps with gene expression

what is a DNA microarray

technique for large scale analysis of gene expression that uses isolation of mrna to prepare cdna in the presence of fluorescent dNTPs

what is rna seq

used for large scale technique of gene expression.rna fraction of interest is selected, fragmented and reverse transcribed. it is sequenced, mapped onto genome and quantiated

what is gene knockdown?

happens via RNA interference pathway

what is gene disruption and genome editing techniques?

in homologous recombination, the target gene is replaced with a mutant version of the gene. genome editing involves a double-stranded break in DNA followed by a faulty repair mechanism

what is the crispr cas-9 system

genome editing technique where a single guise RNA compleof dna mentary to the target sequence directs the site specific cleavage of cas9. requires a pam sequence adjacent to the target dna

what direction is the template strand of DNA read in by RNA Polymerase?

3’ to 5’ direction

what direction is the RNA strand synthesized in by RNA Polymerase?

5’ to 3’ direction to produce a complementary strand of mRNA

what determines the transcription start site?

promoter sequences that lie upstream of the initiating nucleotide

what are promoters?

they are binding sites for RNA polymerase

what is needed for termination of transcription for prokaryotes?

terminator sequence that forms an RNA hairpin or a protein factor called rho that has an RNA/DNA helicase activity

what is needed for termination of transcription for eukaryotes?

RNA pol. II which involves mRNA cleavage and polyadenylation

what is the process for eukaryotic mRNA transcripts?

they are modified with a 5’ cap (7-methylguanosine) and a 3’ poly(A) tail. introns are removed by spliceosome and mature mRNA transcript is competent for translation

what makes up a tRNA?

an amino acid attachment site at one end of the molecules and an anticodon that binds to the mRNA at the other end

how is genetic code read?

as a nucleotide triplet where it is nonoverlapping, degenerate, and nonrandom

what are ribosomes?

large multisubunit complexes that consist of proteins and rRNAs

where does the ribosome bind to in prokaryotes for translation?

shine-dalgarno sequence upstream of AUG

where does the ribosome bind to in eukaryotes for translation?

the 5’ cap of the mRNA and starts at the first AUG codon

when is translation terminated?

when the ribosome reaches a stop codon and binds to a release factor in place of tRNA, resulting in hydrolysis of the petidyl-tRNA and release of the polypeptide

when can gene expression be regulated?

at any step from transcription initiation to protein degradation

what is the most common and powerful way of regulating gene expression?

control of transcription

what can affect the binding of RNA polymerase to promoters and the accessibility of DNA to the transcriptional machinery?

transcriptional activators and repressors

what are the most abundant and structurally diverse type of biological molecule?

carbohydrates

what do carbohydrates consist?

carbon, hydrogen, and oxygen

what are the building blocks of carbohydrates?

monosaccharides

what are monosaccharides?

small molecules containing three to seven carbons that are bound to hydroxyl groups and have varying stereochemistry configurations and can be linked together to form a large variety of oligo and polysaccharides

what are the biological functions of carbohydrates?

energy sources, structural components, cell-to-cell recognition and communication

how are monosaccharides classified?

aldoses or ketoses and can be classified based on the number of carbons they contain

what are isomers?

they have the same molecular formula but different structures

what are constitutional isomers?

differ in the order of attachment of atoms

what are stereoisomers?

atoms are connected in the same order but differ in spatial arrangement

what are enantiomers?

nonsuperimposable mirror images

what are diastereoisomers?

isomers that are not mirror images

what are epimers?

differ at one several asymmetric carbon atoms

what are anomers?

isomers that differ at a new asymmetric carbon atom formed on ring closure

why is it more favorable for the bulky OH groups to be in the equatorial positions?

it avoids steric clash

what conformation does furanose adopt?

they adopt the envelope which is C-2 or C-3 out of the plane of the ring

what kind of bonds does carbohydrate form?

an o-linked and n-linked glycosidic bonds

what is an o-linked glycosidic bond?

when the anomeric carbon reacts with an alcohol group and are found in the polymeric forms of carbohydrates and in glycoproteins

what is an n-linked glycosidic bond?

it is formed when the anomeric carbon reacts with an amine group and are found in nucleotides and nucleic acids as well as in glycoproteins

how is a disaccharide formed?

when two monosaccharides undergo a condensation reaction that results in the formation of an O-linked glycosidic bond

what are storage polysaccharides?

starch and glycogen

what is starch?

in plants, this is where glucose is stored which consists of a-amylose and amylopectin

what is glucose stored in animal cells?

glycogen, which is similar in structure to amylopectin but has more branch points

what are structural polysaccharides

cellulose and chitin

what is the major structural component of plant cell walls?

cellulose which is linear (unbranched) polymer of glucose residues that are joined by B(1-4) linkages

how is cellulose formed?

through sheet-like structures that are held together by a network of hydrogen bonds

what is the primary structure of invertebrate exoskeletons

chitin, it is a linear polymer of N-acetylglucsamine residues that are joined by B(1-4) linkages

what does amylose favor?

a(1-4) linkages which favors the formation of a more compact helix

what type of linkage does a carbohydrate have on a serine or threonine residue to form a glycoprotein?

o-linked