DNA: base, sugar, phosphate

Purines (A and G) vs. Pyrimidines

Bases only connect to sugar

Phosphate connects the sugars to each other; 3' Cconnects to phosphate connecting to 5' C

Isolated nucleic acidds from different organisms-->

Amount of A was almost always the same as the amount of T; same for G and C

A-T has 2 H bonds; G-C has 3 H bonds

10 bp per turn of DNA

Major Groove and Minor Groove (certain proteins bind)

Used chargaff's rule to find that DNA forms a double helix.

Rosalind Franklin- worked in same lab as Maurice Wilkins and used x-ray diffraction to study wet fibers of DNA (diffraction pattern interpreted).

Genome-sum of all information in the DNA

Simpler organisms have smaller genomes, and more complex organisms have larger genomes (the number of genes might not matter necessarily, but what our bodies do with those genes).

Bacteria have 1 few million bp; Human have 3 billion bp

RNA/DNA, SS or DS, Linear or Circular, 1000 to 100,000 nucleotides.

Viruses with a simple structure can self assemble; genetic material and capsid proteins spontaneously bind to each other (tobacco mosaic virus)

Nucleoid (fixed into cytoplasm)- not floating

Loop Domains- compacts chromosome 10-fold

DNA wants to exist at 10 bp per turn- if molecule is wound too tightly or loosely, it will react in a way to restore its balance.

Underwinding and overwinding can induce supercoiling.

Fewer turns is negative supercoiling (underwinding) and more turns is positive supercoiling (overwinding).

Negative Supercoiling induces strand separation for replication at ORI.

Sequence Complexity- number of times a particular base sequence appears in the genome.

Long, linear DNA; Landmark sequences (ORI), Structural genes, Repetitive regions (telomeres).

Repetitive- unique or non-repetitive (structural genes), moderately repetitive (rRNA genes), highly repetitive (ALU, transposons)

Highly repetitive takes less time than moderately repetitive DNA and unique DNA to reassociate after denaturing.

DNA-Protein complex (chromatin)- DNA is wrapped around histones (nucleosome)-optomer is 8 histone proteins bounded by DNA ("beads on a string")- H1 caps DNA on nucleosome

H1 binds nucleosome where the DNA enters and exits the core; H1 may participate in forming higher order structures

modification of histone tails loosens or tightens histone association with DNA (amino acids available for chemical modification)-30nm fiber forms in zigzag pattern of nucleosomes-30nm fiber attaches to scaffold, generating loops

Euchromatin- transcriptionally active, loosely packaged (during interphase-DNA replication)

Heterochromatin-transcriptionally inactive, tightly packed (during metaphase)

Constitutive Heterochromatin-permanently inactive for transcription, always heterochromatin, contain highly repetitive sequences (structural)

Facultative Heterochromatin- regions that can interconvert between euchromatin and heterochromatin.

Metaphase is the most condensed time of chromosomes (no risk of chromosomes getting mixed up during cell division).

1) Binding of DnaA to AT-rich regions

2) Binding of Helicase (unwinding)

3) Single Stranded Binding Proteins prevent secondary structure (folding on itself)

4) DNA Gyrase (topoisomerase) relieves supercoiling (introduces negative coils to relax overwound region). Breaking and re-linking the DNA backbone in order to eliminate extra turns. (requires energy).

5) Primase makes short RNA primer.

6) DNA Polymerase 3 in 5' to 3' direction( DnaP adds to existing strand)

7) DnaP is a complex of 10 proteins (holoenzyme) makes a "clamp" subunit (efficient and stable)

8) Addition of nucleotides 5' to 3'

Incoming nucleotide (a deoxyribonucleoside triphosphate) comes in and the two phosphate groups break off allowing the phosphate to bind to the 3'C of the sugar at the 3' end (ester bond).

TUS proteins bind TER regions which stop helicase

Decatenation of Daughter DNAs (topoisomerase can cut a DNA backbone and unlink two circles) separates the parent and daughter circles of DNA.

Base Pairs prefer to bind correctly (A-T and G-C is most stable); mismatch causes DnaP to pause, leaving mismatched nucleotide near the 3' end (exonuclease- chew back in the 3' to 5' direction to fix mistake).

Exonuclease: 3' end enters the exonuclease site-strand is digested in the 3' to 5' direction until incorrect nucleotide is removed

More DNA to copy, tightly packed, highly regulated cell cycle, linear chromosomes.

Multiple ORI (bacteria have only one) for replication; not believe that there is a specific initiation sequence of DNA (most likely has to do with packing)

Alpha- works with primase to make primer

Epsilon-leading strand synthesis

Delta-lagging strand synthesis

Gamma-mitochondrial

Other- DNA damage/repair

Telomeres- repetive sequence at the end of linear chromosome

Telomerase- protein + RNA

TERT- telomerase reverse transcriptase

TERC-telomerase RNA component

When a terminal RNA primer is removed, the sequence cannot be replaced by DNA polymerase (DnaP cannot start DNA synthesis)- telomerase synthesizes a 6-nucleotide repeat (replaces telomere sequence)

Active Telomerase

Cancer- most human cancer cells reactivate telomerase (inactive in most human somatic cells); Aging-association with the depletion of chromosome ends and aging

Reasearch: If we prevent telomerase gene expression in cancer cells, will it slow cancer cell growth? --> this RNAi specifically binds to telomerase mRNA and prevents translation (slows cancer growth)

Nucleotides (dNTPs), purified Polymerase, Primers, Template DNA (heat up DNA to separate strands since we don't have helicase), buffer/salt (pH)

In Vitro E. coli Experiment: Template DBA, E. coli extract, labeled dNTPs (to be sure it was actually made in the tube)

Structural Genes- 90%; will be translated into protein

Non-Structural Genes- Will not be translated; RNA will serve function

DNA: regulatory proteins bind (influence rate of transcription), promoter (recognition site for transcription factors- causes RnaP binding-initiates transcription), terminator (signals end of transcription)

Deoxyribose is missing O at the 2' C

Nucleoside= Sugar and Base

Nucleotide= Sugar, Base, and Phosphate Group

Upstream<--Regulatory Sequence on mRNA (coding strand) --- -35 sequence starts promoter region --- -10 has TATA box ---  +1 starts transcriptional site --> Downstream

Correlation to consensus determines "strength" of promoter

sigma factor recognizes promoter and RnaP holoenzyme forms closed complex-an open complex is formed and a short RNA is made- sigma factor is released and the core enzyme is able to proceed down DNA

(RnaP, sigma factor, core enzyme COMPLEX)

DNA strand known as the Template Strand (non-coding strand) is used to make a complementary copy of RNA as an RNA-DNA hybrid

RnaP moves along template strand in 3' to 5' direction, and RNA is synthesized in 5' to 3' direction using nucleoside triphosphates as precursors.

Rho (p) dependent termination: Rho protein travels along RNA, hairpain forms (Rho recognition site), Rho causes dissociation of RnaP and transcript from DNA (terminator sequence of G-C's)

Rho (p) independent termination: NusA protein holds RnaP in place, stem loop forms (A-U-rich region), transcript and protein disassociaties.

Stem loop forms and allows polymerase to pause.

RnaP 1- rRNA genes (nonstructural genes; components of ribosome)

RnaP 2- mRNA genes (all structural genes)

RnaP 3- tRNA genes (translation-helps bring right amino acid in the right order to build protein)

Promoter sequences are more variable and often more complex than bacteria.

Structural genes contain in promoters: regulatory elements, TATA box (specifies where RnaP should start), Transcriptional Start Site

Enhancers (increase trancription rate) and Silencers (decrease transcription rate)- can be close to gene or really far away upstream.

Cis-acting elements: DNA sequences that exert their effect only over a particular gene (TATA box, enhancers/silencers)

Trans-acting elements: Regulatory proteins that bind to such DNA sequences (protein could be made on entirely different DNA sequence, but binds to a particular gene- act on the upstream elements).

3 proteins required for basal (normal) transcription to occur at promoter: RnaP 2, Five different GTFs (general transcription factors, and a protein complex called mediator

GTFs bound by activators (increase transcription) and repressors (decrease transcription).

TF2D binds to the TATA box- inculde TATA-binding proteins (TBP) and several TBP-associated factors (closed complex).

TF2H: contains helicase (open complex-allows unwinding to occur).

After open complex is formed, other GTFs are released.

phosphorylates CTD domain of RnaP and it may regulate the ability of TF2H to phosphorylate the CTD- RNA can now proceed to elongation

(promotes more activation of polymerase or less)

RnaP transcribes a gene past the polyA signal sequence- RNA is cleaved just past the polyA signal sequence

Allosteric model (change shape due to binding): RnaP2 is destabilized due to the release of elongation factors or the binding of termination factors.

Torpedo Model: An exonuclease binds to the 5' end of the RNA that is still being transcribed and degreades it in a 5' to 3' direction (exonuclease catches up to RnaP 2 and causes termination).

mRNA have a string of Adenine nucleotides at their 3' ends; not encoded in the gene sequence- added enzymatically after the gene is completely transcribed

Polyadenylation signal sequence (consensus sequence in higher eukaryotes)- endonuclease cleave occurs about 20 nucleotides downstream from the AAUAAA sequence and PolyA-polymerase adds adenine nucleotides to the 3' end

adding 7-methylguanosine to 5' end of mRNA-recognized by cap-binding proteins (proteins will recognize the cap-binding proteins and help transfer it from the nucleus to the cytoplasm where it will be translated- protects RNA)

RNA 5'-triphosphates removes a phosphate and Guanylyltransferase hydrolyzes GTP and the GMP is attached to the 5' end and PPi is released (added right away during elongation)- methyltransferase attaches a methyl group

Transcription produces entire gene product: introns are removed later and exons are spliced or connected together.

mix together denatured DNA and mature mRNA- the mature mRNA binds to template DNA strand, which causes the intron DNA (which has been spliced out of the mRNA) to loop out and mRNA cannot hybridize-->beta-globin gene contains introns

Group 1: Free guanosine bound to site IN intron breaks the bond between exon 1 and intron and becomes attached to 5' end of intron-->results in exon 1 and 2 covalently joined and free

Group 2: 2' hydroxyl from adenin WITHIN intron breaks bond between exon 1 and intron--> results in Exon 1 and 2 linkage and intron as lariat (

snRNPs (small nuclear RNA and a set of proteins) bind to an intron sequence and precisely recognize intron/exon boundaries-Hold the pre-mRNA in the correct configuration-Catalyze the chemical reactions that remove introns and covalently link exons (phosphodiester linkage)

snRNPs bind to each consensus region and loop out the introns so thta the exons come together (introns loop out, are cut, and exons are connected)

Transfer RNA- cleaved at both the 5' and 3' ends to produced mature, function tRNAs (exonucleases-RNaseD- cleaves a covalent bond b/w 2 nucleotides at one end of a strand and endonucleases-RNaseP- cleaves bond within a strand)

RNase P is a ribozyme (RNA + protein)

processing of rRNA occurs in nucleolus and produces multiple mature rRNA transcripts (key in ribosome structure)