Ch. 18,21 : Gene Expression: The genetic code & Transcriptio

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Gene expression
-central dogma of molecular bio:

the use of info in DNA to direct the production of proteins
-DNA-RNA-protein

Nuclear env protects

chromo and immature RNA from cytoplasmic enzymes
Nuclear pores
direct continuity between cytoplasm & nucleoplasm
-mammalian nucleus ~3,000-4000 pores
-eukaryotic cells must transport macrmolecues in & out of nucleus
Where does replication and trancrption occur? and where does translation occur?
-replication and transcription in nucleus, translation in cytosol
Nuclear pores
-Inner & outer membranes nuclear env fuse at nuc pore complex with intricate protein structure
-small molecules (<10nm) diffuse through nuc pores (dNTP's, ions, small proteins-histones)
Transcription occurs in the
Nucleus (eukaryotes)
Cytosol (prokaryotes
A cell uses 3 kinds of RNA to make proteins
messenger RNA (mRNA)
transfer RNA (tRNA)
ribosomal RNA (rRNA)
Overview of Transcription:
Binding of RNA polymerase to DNA promoter sequence
Initiation of RNA synthesis- base pair w/DNA template
Elongation of RNA strand
Termination at termination signal, release RNA molecule, RNA polymerase dissociates from DNA molecule
Binding- RNA polymerase binds at DNA promotor site
-the transcriber is RNA polymerase
-consists of 2 alpha, beta and beta prime and sigma factor
- complete holoenzyme- ^ required to ensure initiaiton at proper DNA site
-Sigma factor ensures correct binding of RNA poly to promotor
Promoter
is upstream (neg #), before transcribed sequence/gene- downstream (pos #), no # "0"
-specific sequence, determines which strand to serve as template
-statr point (+1) described 5'-3' on coding strand (opp template strand)
-not identical, have consensus sequences (most common)
-mutations interfere w/promoter fct/activity
-triggers local unwinding of DNA strands by topoisomerase

INITIATION

-link up dNTP's in 5'-3' direction to make RNA strand
-RNA-DNA hybrid- complementary base pairing to DNA template
-Add until ~9 nucleotides long
ELONGATION
-RNA poly core enzyme (2 alpha,1 beta,1beta prime, prime) reads along the DNA, untwist DNA double helix
-Adds nucleotides to RNA strand
-Proofreading- RNA poly has 3'-5' exonuclease activity-correct mistakes-remove wrong nucleotides,insert correct bases

TERMINATION- reach termination signal
type 1 of 2:

1-Need rho (p) factor
-binds to specific 50-90 base termination sequence
-needs ATP to unwind & release new RNA from DNA template
-Release RNA poly
: RNA poly can bind sigma factor, start another RNA synthesis event
type 2 of 2:
2-Does not need rho (p) factor
-RNA has short complementary sequence to each other & short GC-rich sequence, w/several U's
-RNA forms hairpin loop
-pulls RNA away from DNA
: AU bonds- weaker

Differences in Eukaryotic Transcription
Protein-protein interactions

-Need transcription factors (TF)- must bind to DNA before RNA poly can bind to promoter
-Bind to other proteins & DNA
-Recruit coactivator proteins- help assymbly of RNA poly complex to initiate RNA syn
-Unwinds DNA
Elongation (euk.) need special proteins to dissassemble nucleosome in front of RNA poly & reassemble behind RNA poly
Termination (Euk)
-many signals depending on type of RNA poly
-short run of U's
-RNA cleavage:
special AAUAAA signal - cut 10-13 bases downstream, then add poly(A) tail at cleavage site
-RNA processing
Ribosomal RNA -the most abundant (70-80%), most stable RNA in cells- component of ribosomes
Transfer RNA - 10-20%- critical in protein syn
Messenger RNA -less than 10%- amino acid sequence
rRNA
-single primary transcript unit with transcribed spacers
-150-200 copies in cell
-Methylation of ribose sugars by small nucleolar RNA's (snoRNA's)
-series of cleavage reactions to release mature rRNAs
-transcribed spacers are degraded
-chemically modify euk primary transcript into mature RNA
Methylation addition of methyl groups-protect from enzyme degredation (esp rRNA) then transport to cytosol for translation
5' methyl guanine cap
to 5' end of pre-mRNA & mRNA
-added after initiation of RNA syn
-makes RNA more stable so cannot be degraded
-position of ribosome to start translation
Poly (A) tail
50-250 nucleotides of A's added to 3' tail
-NOT FROM DNA SEQUENCE-found in termination signal
-added by poly (A) polymerase after special AAUAAA signal
-make it more stable, longer life span
-help export mRNA from nucleus to cytoplasm
-help ribosome recognize mRNA to be translated
Introns
-intervening sequences within the euk primary transcript absent in mature functional RNA- not expressed
-can be 99% of genes DNA
RNA spliced...
removes introns, joins exons (expressed sequences)
-by ribozymes- self-splicing RNA introns
-by spliceosomes- RNA-protein complex
Catalyzed by spliceosome -
RNA & protein complex (small nuclear ribonucleoproteins, snRNPs)
-recognizes 5' to 3' splice sites-cut & link exons

Why do eukaryotic genes have introns?
1 of 5

-generate numerous diff mRNA's from 1 RNA transcript through alternative RNA-splicing-exon shuffling
2 of 5
-help increase biological complexity increase in gene number (25,000 genes produce 200,000-1 million proteins
-ex: immune system
-bacteric and archaea lost introns from their streamlined genomes
3 of 5 Evolutionary- hasten evolution of new and potentially useful proteins
4 of 5 each exon codes for diff fct'l regions- protein can fold independently into separate domain
5 of 5
Nucleic acid editing- more 1-100's nucleotides inserted or converted
-can inactivate retroviruses
-ex. HIV has defense protein- destroys DNA-editing enzyme
Translation
converts nucleotides of a gene into amino acids in a protein in order using genetic code
-almost universal- prokaryotes, euk, & viruses
-established early on in history of life on Earth
(translation) mRNAs are...
"read" by ribosome in 3-nucleotide units, termed
(64 possible combinations)
-each codon codes for an amino acid or stop signal
-code is degenerate- adaptibility, some are silent mutations
Exceptions- few slight differences (translation)
-In mitochondria, a few bact & unicellular org's
-UGA-21st amino acid selenocysteine, UAG-22nd amindo acid-pyrrolysine