biochemistry molecular biology high yield

Base sequence recognized at origin of replication Helicase breaks hydrogen bonds holding base pairs together → unwinding begins Single-stranded DNA binding protein SSB binds to each s

Nucleic Acid Components

•FA 2013: 64.2 • FA 2012: 68.2 • FA 2011: 67.1

BC01_1-

Structural elements of the most common nucleotides

• Nucleoside: nitrogenous base + ribose

• Nucleotide: nucleoside + phosphate group(s)

• Nucleic acids: polymers of nucleoside monophosphates

Nucleic Acid Components

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BC01_1- 3

•FA 2013: 64.2 • FA 2012: 68.2 • FA 2011: 67.1

• ME 3e: 60.3 • ME 4e: 60.3

BC01_1-

• Phosphate group links 3’ carbon of

deoxyribose to 5’ carbon of another

Total purines = pyrimidines

• Strands are anti-parallel 5’ to 3’

Nucleic Acid Composition

BC01_1- 4

•FA 2013: 64.2 • FA 2012: 68.2 • FA 2011: 67.1

• ME 3e: 60.3 • ME 4e: 60.3

• Negatively charged DNA

winds around positively

charged histone octamer

• Histone octamer: 2 copies of

BC01_1-

Euchromatin

• Loosely condensed nucleosomes

• Accessible for transcription

Heterochromatin

• Densely condensed nucleosomes

• Inaccessible for transcription

Chromosome Composition

BC01_1- 6

•FA 2013: 64.1 • FA 2012: 68.2 • FA 2011: 66.1

• ME 3e: 60.3 • ME 4e: 60.3

BC01_2-

Definition

• Process by which genetic information is

transmitted from cell to cell

Process

• 2 complementary strands of parental

DNA are pulled apart

• Each is used as template for synthesis

Base sequence recognized at origin of replication

Helicase breaks hydrogen bonds holding base pairs

together → unwinding begins

Single-stranded DNA binding protein (SSB) binds to

each strand

Primase synthesizes RNA primer (5’ → 3’) at origin of each parental strand to be complementary and anti- parallel to DNA template strands

DNA polymerase (III in prokaryotes and α/δ in eukaryotes) begins DNA synthesis

Leading strand: continuous and toward replication fork Lagging strand: Okasaki fragments, away from

replication fork RNAse H removes primers, DNA polymerase fills gaps

DNA polymerase has capacity to proofread by possessing 3’-exonuclease activity

DNA ligase seals nicks between Okasaki fragments

Topoisomerase II (DNA gyrase) relieves strain of

supercoiling Replication is completed when 2 forks meet

DNA Replication

Site of quinolones, etoposide action !

BC01_2-

Telomeres

• Repetitive sequences at ends of linear DNA molecules

• Replication shortens telomere length, contributing to genetic

material loss and cell aging

Telomerase

• Enzyme which maintains telomere length (embryonic, germ,

and stem cells) by replacing telomere sequences on parent

strand

• Inactive in somatic cells (skin, blood, connective tissue)

• Prokaryotes have single circular chromosomes, hence no

1 Nucleotide excision repair

phosphodiester backbone of damaged strand

• Removes defective oligonucleotide

• DNA polymerase fills gap & DNA ligase seals

• Skin cancer (melanomas)

• Diagnosis: excision endonuclease levels

BC01_1-

2 Base excision repair

• Example: cytosine deamination (loss of amino group leads to conversion of cytosine into uracil)

• Glycosylases and AP endonucleases recognize and remove damaged bases

3 Mismatch repair

• System for recognizing and repairing DNA replication errors Main genes

involved in mismatch repair are hMLH1 and hMSH2

Hereditary nonpolyposis colorectal cancer (Lynch syndrome)

• Mutation in genes encoding for mismatch repair enzymes → cancer

• ↑↑ risk for colon, endometrial, ovarian, and gastric cancers

• Example of incomplete penetrance

DNA Repair

BC01_3- 2

•FA 2013: 69.1 • FA 2012: 73.1 • FA 2011: 71.1

• ME 3e: 64.2 • ME 4e: 64.2

DNA Repair Mechanisms Summary

BC01_3- 3

•FA 2013: 69.1 • FA 2012: 73.1 • FA 2011: 71.1

• ME 3e: 64.2 • ME 4e: 64.2

BC01_4-

• First stage in “expression” of genetic information

• Information from double-stranded DNA molecule transcribed into base

sequence of single-stranded RNA molecule

• Only one strand of DNA molecule (template strand) is read and the other strand (coding strand) is not

• Main enzyme is RNA polymerase (reads in the 3’→ 5’ direction, synthesizes RNA molecule in 5’ → 3’ direction)

• End product is complementary, antiparallel, single-stranded RNA molecule

Overview of Transcription

BC01_4- 1

•FA 2013: 70.3 • FA 2012: 75.1 • FA 2011: 72.3

• ME 3e: 65 • ME 4e: 65

BC01_4-

• Structural component of ribosomes

• Most rRNA made in nucleolus

• Carries amino acids to ribosome

• Most tRNA made in euchromatin portion of nucleus

• Carries information specifying amino acid sequence of a protein to ribosomes

• Only type that is “translated” into proteins

• Different mRNA molecule for each of thousands of proteins

• Most mRNA made by euchromatin portion of nucleus

BC01_4-

Protein factor ῥ required for transcription termination

Transcription factors help initiate transcription

RNA Polymerases

Single RNA polymerase

RNA polymerase 1: rRNA (nucleolus) RNA polymerase 2: mRNA (nucleoplasm) RNA polymerase 3: tRNA (nucleoplasm)

RNA polymerase

Actinomycin D inhibits transcription in eukaryotes

and prokaryotes

BC01_4- 3

•FA 2013: 70.5 • FA 2012: 75.3 • FA 2011: 72.5

• ME 3e: 65 • ME 4e: 65

BC01_4-

• Promoter region: binding site for RNA polymerase

• No primer required

• RNA polymerase reads template strand of gene and moves 3’ → 5’ direction

• RNA polymerase synthesizes 5’ → 3’ direction

• No proofreading

• mRNA sequence identical to DNA coding strand (uracil instead of thymine)

• First nucleotide transcribed defined as +1 nucleotide

• Transcription ends when RNA polymerase reaches termination signal

Transcription Terminology

BC01_4- 4

•FA 2013: 69.2 • FA 2012: 74.1 • FA 2011: 71.2

• ME 3e: 65 • ME 4e: 65

BC01_4-

• Genes are composed of exons and

introns

• Both are transcribed but introns

removed during RNA processing

• Mature mRNA molecules translated

into proteins by ribosomes in the

cytoplasm

• RNA polymerase II:

Recognizes and binds to promoter

region (TATA and CAAT box) with help

from transcription factors

Separates DNA strands to initiate

transcription

Ends transcription when reaches

termination signal

The pre-mRNA (primary transcript)

undergoes co- and postranscriptional

BC01_4-

• 7-methylguanosine cap added

to 5’ end

• Poly-A tail attached to 3’ end

• Introns removed by splicing

• Excised introns (lariat)

degraded in nucleus

• Exons joined together

• Mutations of 5’ and 3’ splice

sites of introns affect accuracy

of intron removal

• Mutations may lead to introns

being left within mature mRNA

Co- and Postranscriptional Processing

SLE patients make

BC01_4-

• Production of 2 or more proteins

variants from the same gene

BC01_4-

tRNA

sequence

Requires 2 high energy bonds from ATP

Transfers activated amino acid onto 3’ end of correct tRNA

• FA 2012: 77.2 • FA 2011: 74.1 • FA 2010: 74.1

BC01_4-

Definition

nucleotides in DNA and sequence of

amino acids in proteins

Characteristics

61 for AA

3 stop codons (UGA, UAA, UAG)

“AUG” always first codon (methionine)

BC01_4-

Definition

• Permanent heritable change in DNA base sequence

• May be cause of genetic disease, change in enzyme activity, and other properties

of cell function

Mutations

ATG GCA ATT TTA CCT

ATA

ILE

ATG GCA ATT TT G CCT

ATA

ILE

ATG GCA ATT T C A CCT

ATA

ILE

ATG GCA ATT T G A CCT

MET ALA ILE STOP

ATA

ATG GCA ATT TAC CTA

BC01_1-

Definition

• Decoding of mRNA by ribosome to

produce amino acid chain

BC01_1-

• Small ribosomal subunit binds to mRNA

30S small subunit binds to Shine-Dalgarno sequence (prokaryotes)

40S small subunit binds to 5’ cap structure (eukaryotes)

• Charged initiator tRNA binds to start codon

• Large subunit binds to small subunit forming initiation complex

• Ribosomal binding sites

Peptidyl site (P site)

Aminoacyl site (A site)

Translation: Initiation Phase

Small ribosomal subunit

5’ cap

fmet-tRNA (Pr)

Large subunit

BC01_1-

Two ATPs for charging tRNA and peptide bond formation

Two GTPs for 1 st and 3 rd steps of the cyclic sequence

Translation: Elongation Phase

BC01_5- 3

•FA 2013: 73.1 • FA 2012: 78.1 • FA 2011: 75.1

• ME 3e: 66.3 • ME 4e: 66.3

BC01_1-

Translation: Termination Phase

• Occurs when any of the 3 stop codons of RNA moves into A site

• Peptidyl transferase + release factor hydrolyzes completed protein from final tRNA in P site

• Upon completion, mRNA ribosome, tRNA, assisting factors are reused for further protein synthesis

• Multiple ribosomes can bind to single mRNA (polysomes)

BC01_1-

Primary

• Sequence of amino acids

Secondary

• Amino acid chain folded into

energetically stable structure (

α-helix, β-sheet)

Tertiary

• Positioning of secondary structures

in relation to each other to generate

3-dimensional shape

Quaternary

• Interaction among multiple subunits

in proteins (e.g Hemoglobin)

Chaperones, Ubiquitin and Proteasomes

• Microbial peptide fragments can be

presented in antigen presenting cells

BC01_7- 2

•FA 2013: 73.2 • FA 2012: 78.2 • FA 2011: 75.2

• ME 3e: 67.1 • ME 4e: 67.1

Smooth endoplasmic reticulum (SER)

Nucleus

Golgi apparatus Lysosome

Peroxisome Mitochondrion

Microtubule

& filament

RER ribosome

Golgi apparatus

RER

Free ribosome

BC01_7- 3

•FA 2013: 74.2 • FA 2012: 79.2 • FA 2011: 76.2

• ME 3e: 67.3 • ME 4e: 67.3

• Disulfide bond formation

• N-Glycosylation and/or O-glycosylation

• Proteolysis

• Phosphorylation

• Gamma carboxylation

• Prenylation

N-terminal hydrophobic signal sequence

• Required on proteins destined to be:

Secreted outside of cell (e.g., insulin)

Co- and Post-translational Modification of Proteins

BC01_7- 4

•FA 2013: 73.2 • FA 2012: 78.2 • FA 2011: 75.2

• ME 3e: 67.3 • ME 4e: 67.3

BC01_1-

Golgi Apparatus

Definition

• Organelle that functions as modification and

distribution center for proteins and lipids

BC01_1-

Primary structure

composed of

tripeptide Gly-X-Y

Post-translational Modification of Collagen

Synthesis of pre pro- α chain with N-terminal

hydrophobic signal sequence

Removal of signal sequence by signal peptidase

Hydroxylation of selected

prolines and lysines using

vitamin C

Glycosylation of selected hydroxylysines

Triple helix formation (procollagen)

Secretion from cell (pro-collagen secreted)

Cleavage of pro-peptides

Assembly into fibrils Stabilized by lysyl oxidase (Requires O2 and Cu + )

Aggregation to form a collagen fiber

Rough endoplasmic reticulum

II

Thin fibrils Structural Cartilage Vitreous humor - -

III

Thin fibrils Pliable Blood vessels Granulation tissue Ehlers-Danlos type IV Keloid formation

IV Amorphous Basement membranes

Goodpasture syndrome

Alport disease Epidermolysis bullosa

BC01_8- 2

•FA 2013: 77.2 • FA 2012: 82.3 • FA 2011: 79.2

• ME 3e: 68.2 • ME 4e: 68.2

BC01_1-

Disorders of Collagen

Scurvy Deficient hydroxylation secondary to ascorbate

deficiency

Petechiae, ecchymoses, loose teeth, bleeding gums, poor wound healing, poor bone development

Osteogenesis

Imperfecta Mutations in collagen genes

Skeletal deformities, fractures, blue sclera

Menkes Disease Deficient cross-linking secondary to copper deficiency

Depigmented (steely) hair, arterial tortuosity and rupture, cerebral degeneration,

osteoporosis, anemia

BC01_8- 3

•FA 2013: 78.3 • FA 2012: 83.3 • FA 2011: 80.1

• ME 3e: 68.2 • ME 4e: 68.2

BC01_1-

General characteristics

• Membrane enclosed

• Spherical organelle

High Yield functions

• Synthesis and degradation of H2O2

BC01_1-

High yield functions

• Detoxification of drugs and

BC01_1-

• Hollow tubes composed of polymerized dimers of α and β tubulin

• Tracks for intracellular transport

• Specific ATPase motor molecules

Dynein: retrograde transport

Kinesin: anterograde transport

Location

• True cilia and flagella

• Mitotic spindle (during mitosis/meiosis)

Drugs acting on microtubules

BC01_1-

• Autosomal recessive

• Microtubule polymerization defect

→ impaired fusion of phagosomes

BC01_1-

Structure

• Nine peripheral pairs of microtubules

• Two central microtubules

• Immotile spermatozoa → infertility

• Chronic respiratory infections

BC01_1-

Actin and myosin

• Components of muscle sarcomeres, microvilli, adherence junctions

Desmin (skeletal, cardiac, and GI tract smooth muscle cells)

Vimentin (fibroblasts, fibrocytes, endothelial cells, vascular smooth muscle) Glial fibrillary acidic protein (astrocytes, Schwann cells)

Peripherin (peripheral nerve axons)

• Type III: neurofilaments (neurons)

• Type IV: lamins (form meshwork inside nuclear envelopes of all cells)

Cytoskeletal Structures

BC01_9- 6

•FA 2013: 76.3 • FA 2012: 81.4 • FA 2011: 78.3

• ME 3e: 81.1 • ME 4e: 81.1

Megan Murray M.D., Ph.D Candidate University of Buffalo

Biochemistry 2

Molecular Biology Part II

• DNA packaged in chromatin structures

• Gene expression requires chromatin

remodeling

Transcription factor (proteins) bind to specific

DNA sequences and recruit other coactivators

Histone acetylase, acetylates lysyl residues on

histones to neutralize their positive charges,

weakening interactions between histones and

DNA

Remodeled chromatin has more exposed DNA

for additional transcription factors and other

proteins to bind and initiate transcription

Chromatin Remodeling

BC02_1- 2

•FA 2013: NA • FA 2012: NA • FA 2011: NA

• ME 3e: NA • ME 4e: NA

Characteristics

• DNA sequences that to which specific transcription factor proteins bind

• May be located upstream, downstream, or within an intron of the gene

• Can act in a tissue-specific manner

• Multiple DNA sequences called response elements may be grouped within

the enhancer region

Enhancers

BC02_1- 3

•FA 2013: 70.3 • FA 2012: 75.1 • FA 2011: 72.3

• ME 3e: NA • ME 4e: NA

Definition

• Activator proteins that bind to response elements within enhancer regions

Domains: 2 within a transcription factor

• DNA binding domain

Zinc fingers ( e.g steroid hormone receptors)

Leucine zippers (e.g., cAMP dependent transcription factors)

Helix-turn-helix (e.g., embryonic gene expression transcription factors)

• Activation domain, which allows

Binding to other transcription factors

Interaction with RNA polymerase

Recruitment of chromatin modifying proteins

Transcription Factors

BC02_1- 4

•FA 2013: NA • FA 2012: NA • FA 2011: NA

• ME 3e: 65 • ME 4e: 65

Stimulation of Transcription

General transcription factor

(example)

Specific transcription factor (example)

DNA binding domain

Activation domain

Types of Transcription Factors

• General: bind to DNA sequences within the promoter, allowing RNA polymerase to

form initiation complex Found in most genes within most cells Involved in basal

transcription of genes

• Specific: localized to specific cells and tissues Bind to enhancer/silencer regions,

modulating formation of initiation complex

BC02_1- 5

•FA 2013: NA • FA 2012: NA • FA 2011: NA

• ME 3e: 65 • ME 4e: 65