A. P. and M.B. (EXAM 1)

Transcriptome

collection of all RNA transcripts in a celll

Proteome

Collection of all proteins in a cell

What is RPE65 used for

required for processing retinol esters during retinoid(visual) cycle

Gene-based therapy strategies

- replace missing gene product

- stimulate normal &/or block pathological processes

- Eliminate/inactivate toxic proteins

Gene therapy strategy ( missing gene product replacement )

add back biosynthetic component by way of dietary supplement

-intorduce new gene into affected cells

Gene Therapy strategy: stimulate normal &/or block pathological processes

-block proliferation of target cells

- block blood vessel formation and growth

- add survival factors or genes that block apoptosis

mutations that destroy or inactivate the enzymes resulting in pathway blockage can cause

- buildup of toxic substrates

- absence of final required product

General steps of Somatic Celll Gene Therapy

1. Identify mutation in pt

2. clone correct/ normal gene

3. package DNA for normal gene in viral vector

4. Virus infects cells and sends DNA to nucleus

5.NEw gene inserts into genome and makes correct gene product

Solutions that address excess proliferation of cells

-Destroy neovascular tufts (laser)

- reduce blood vessel growth ( block vessel growth signaling pathways

- kill abnormal cells(chemotherapy)

-removal of turmor( surgery)

-block proliferation ( antibiotics)

Solutions that address excess cell death

-eliminate/ inactivate toxic proteins

- add survival factors or genes that block apoptosis

Therapeutic Strategies for neuromuscular retinal disease

-laser ablation (laser off abnormal vessels)

-photodynamic therapy( Verteporfin injection which helps form blood clots to stop leakage )

- block blood vessel growth ( inhibit, block, or destroy VEGFA)

High levels of VEGF-a causes

vasoproliferation: blood vessel formation

vasomigration

vasopermeability: leakiness of fluid and proteins from blood vessels

Aptamers

short RNA nucleotides that bind to VEGF and inhibit VEGF

Soluble 'decoys'

proteins that mimic a binding partner of a target protein resulting in prevention of normal binding partner/receptor thus blocking signaling pathway

central dogma of Gene expression

DNA -> RNA -> Protein

conclusion from Frederick Griffiths 1928 experiment

Characteristics of dead bacteria could be passed to other bacteria by something that remains in the cell extracts

histone

protein molecule around which DNA is tightly coiled in chromatin

an October made of 8 subunits

chromatin is composed of

dna and histones

chromatin is organized into

protein scaffolds;

eukaryotic chromatin is organized into chromosomes even when not condensed mitosis or meiosis

a nucleotide is composed

deoxyribose sugar, phosphate group, nitrogenous base

or

base + phospho-deoxyribose

nucleoside is composed of

deoxyribose + nitrogenous base WITHOUT phosphate

ribose is what type of sugar and what do you need to know about each carbon

5 carbon sugar

C1: attachment site of base

C2: has OH

C3: MUST HAVE OH to allow DNA polymerization

C4: has -H group

C5: attachment site for Phosphate

what are the purine bases

Adenine and Guanine

what are the purine nucleoside

Adenosine and guanosine

what are purine nucleotide triphosphate

dATP and dGTP

what are the pyrimidine bases

Cytosine and Thymine

what are the pyrimidine nucleosides

cytidine and thymidine

what are the purine nucleotide triphosphate

dCTP and dTTP

double ring structure on a base indicates it is a

purine

single ring structure on a base indicates it is a

Pyrimidine

can the percent concentration of a nitrogenous base be more than 50%

no

Chargaff's rule for DNA base pairing

%A=%T

%G=%C

A+T+G+C= 100%

which bonds stabilize DNA structure

Hydrogen bonds between bases

which purine-pyrimidine base pair is most stable

C-G bec it has 3 bonds compared to A-T which has 2 bonds

DNA base pairs are more exposed at which groove and why there

major groove and because regulatory protein preferentially bind there

deoxyribose sugars are joined by

phosphodiester bonds

what must be present in order for DNA replication to occur

OH ; not able to add nucleotide

Where does DNA polymerase get its energy from to make phosphodiester bonds

phosphate groups

how are prokaryote DNA polymerases identifies

with Roman numerals

how are eukaryotic DNA polymerases identified

with letters

direction of DNA synthesis

5' to 3'

5' end of new base is added to 3' end of existing DNA

t/f : DNA replication moves in BOTH directions at the replication fork

true

Primase

synthesizes short RNA oligonucleotides copied from DNA

prokaryote

DNA polymerase III

elongates RNA primers with new DNA

prokaryote

DNA polymerase I

removes RNA at 5' end of neighboring fragment and fills gap with DNA

prokaryote

DNA ligase

connects adjacent fragment

prokaryote

DNA polymerase A

synthesizes short RNA oligonucleotides copied from DNA and adds ~20 bp to 3' end of RNA primer

eukaryote

DNA polymerase D

elongates RNA primers with new DNA

synthesizes leading strand copy

eukaryote

RNAse H

removes RNA at 5' end of neighboring fragment and fills gap

Eukaryote

DNA gyrase

removes supercoiled twists that from when DNA strands are separated by cutting and rejoining the strands

topoisomerase

removes supercoiled twists that form when DNA strands are separated

Histone reassembly require how many and which proteins

2 proteins

CAF-1: assembles histone octomer

PCNA= wrap the DNA around Octobers to form nucleosomes

what happens to histones during DNA replication

histones are removed

telomeres

ends of chromosomes

telomerase

lengthens the telomeres after DNA replication

what happens to telomere if telomerase is not present

telomere becomes shorter after each replication and cell division

what happens to cell if chromosomes lose their telomeres

the cell stops dividing

Bacterial DNA is ______ and replicated from ______ origin

circular ; singular

Eukaryotic DNA is _________and replicated from ________ origins

linear; multiple

Open chromatin diameter

11 nm fibers

Condensed chromatin diameter

30 nm

helicase

separates DNA strands by breaking H bonds

Ran synthesis is from__________but the DNA template is read from ______

5' to 3'

3' to 5'

Does splicing occur in prokaryotes

no

what RNA does RNA polymerase I transcribe

ribosomal RNA

what RNA does RNA polymerase II transcribe

messenger RNA

what RNA does RNA polymerase III transcribe

transfere RNA and small nuclear RNA

TATA box

A DNA sequence in EUKARYOTIC promoters crucial in forming the transcription initiation complex.

also called the Goldberg-Hogness box

Pribnow box

the TATAAT sequence that is often found at the -10 region of PROKARYOTIC promoters

the types of functional RNA

tRNA, rRNA, snRNA, miRNA, siRNA

tRNA

transfer rna

brings the amino acid to the ribosome during protein synthesis (translation)

rRNA

ribosomal RNA; type of RNA that makes up part of the ribosome

snRNA

small nuclear RNA

involved in the processing and modification of DNA and RNA

which RNA regulate gene expression

miRNA

siRNA

miRNA

micro rna

contains complementary sequences to mRNA target

bind to the 5' untranslated region of mRA target

targets RNA doe degradation by cell

siRNA

small interfering RNA

anti-viral mechanism present in lower eukaryotes

blocks initiation of translation/protein synthesis

3 main functions of ribonucleotides

-energy

-phosphate donor

-intracellular signaling

difference between ribose and deoxyribose

ribose has a 2'-OH group and deoxyribose has a 2'-H

5' end of the gene contains

promoter and transcription start site (TSS)

how are bases numbered

relative to the transcription start site designated +1

transcription start site (TSS) +1 codes for `

the first base of the RNA transcript

promoter (in DNA)

-regulates initiation of transcription(determines where transcription begins) but not transcribed into RNA

-located at 5' of the gene BEFORE TSS +1

-contains binding site for RNA polymerase

how are each base of the PROMOTER is numbered?

relative to TSS (+1) in minus direction

Transcription start site

+1 codes for the first base at the 5' end of the RNA molecule

present in both protein coding genes and genes encoding functional RNA

5' UTR

5' untranslated region;

located between +1 and translation start site

regulatory and do not code for protein

ATG/AUG significance

codes for first amino acid of the protein that will be made from the RNA transcript

Sense strand of DNA

The strand of DNA that is read to make mRNA during transcription

- use it directly to predict protein sequence from 5' to 3'

also known as non-template strand

coding strand

the strand of DNA that is not used for transcription and is identical in sequence to mRNA, except mRNA contains uracil instead of thymine

rna polymerase for prokaryotes

binds to the promoter via highly conserved sequences

- located at -10 and -35 relative to TSS

-positions RNA polymerase adjacent to the TSS

how many types of RNA polymerase fo prokaryotes have for all types of RNA

1

rna polymerase is a _________ consisting of 5 subunits plus the _________

holoenzyme

sigma subunit

RNA elongation continues after release of

sigma factor

prokaryotes use which two main mechanisms to terminate transcription

intrinsic

rho dependent

intrinsic termination

PROKARYOTIC

- based on spontaneous formation of specific secondary structure

-occurs when a terminator sequence( filled with G-C) is present in RNA molecule

-forms hairpin loop structure in RNA

-energy independent

Rho-dependent termination

-energy dependent

-contains RUT(rho-utilization) site in rna

-rho protein binds to rut site which destabilizes the RNA-DNA-RNA polymerase complex

in eukaryotic gene, promotes starts at

-1 and goes to the left

transcribed region in eukaryotic gene starts at

+1 and goes to left

do prokaryotic genes have introns

no

describe TBP and TFIID

TBP: TATA binding box

TFIID: transcription factor IID binds to TBP

TBP and TFIID recruit initiation complex which is then positioned at TSS and is ready to begin transcription

eukaryotic RNA processing : CAPPING

step 1

Carbon tail domain is phosphorylated which enables assembly of capping enzyme complex

capping complex adds 7-methylguanosine cap to the 5' end of the RNA molecule

5' cap protects/stabilizes RNA