Lecture 45-58 Flashcards
Transcription
Process of RNA synthesis with DNA template. Catalyzed by RNA polymerase. Begins at promoter and ends at terminator
Trans-acting factors/elements
Usually DNA-binding proteins (ex: TFs) but include some non-coding RNAs. Diffusible and function at multiple sites
Cis-acting factors/elements
Usually DNA sequences that are part of genes
mRNA
Encodes primary AA sequence for proteins (template for translation)
tRNA
Carries AA into catalytic site of ribosomes. Base pairs to mRNA to ensure correct AA sequence
rRNA
Structural components of ribosomes
Primary transcript
Finished RNA molecule beginning at promoter and ending at terminator. Used as mRNA in bacteria, but in eukaryotes undergoes further modification
Template strand
DNA strand used in transcription to synthesize RNA. Equals the reverse complement of the coding strand and the primary transcript
Coding strand
DNA strand that resembles the RNA primary transcript (except T for U)
Prokaryotic promoter
Cis-acting element where RNA pol binds to initiate transcription. Have orientation polarity. Consensus sequences on coding strand:
-35: TTGACA
SPACER
-10: TATAAT
SPACER
Most primary transcripts begin with purine and often with CAT sequence (A = +1)
Gene
Unit of heredity with some function in the organism.Most encode information for a protein and includes the DNA encoding the protein and regulatory elements needed for transcription
ORF (cistron)
Sequence of bases that encodes primary protein sequence
Operon
Coordinately regulated gene clusters. May contain multiple ORFs operating with one promoter and terminator. Yields one polycistronic mRNA
E. coli atp operon = 9 ORFs for ATP synthase
Constitutive promoter
Promoter that is always active with rate of transcription initiation determined by sequence identity to consensus
Strong promoter
Has high sequence identity to promoter consensus. Mutations that move toward consensus increase rate of transcription initiation
Weak promoter
Has several base changes from promoter consensus. Mutations that move away from consensus decrease rate of transcription initiation
RNA pol (E. coli)
Catalyzes transcription in 5’-3’ direction along template strand with similar mechanism to DNA pol. Primer independent. Holoenzyme responsible for initiation and synthesis of first 10 nt. Holoenzyme = a2BB’wo subunits
Core enzyme carries out elongation.
Core enzyme = a2BB’w subunits
No proof-reading (3’-5’) exonuclease. High error rate (balanced by high RNA turnover rate)
o (sigma) subunit
Recognizes promoter. 7 different sigma subunits in E. coli. Sigma 70 binds to most promoters (consensus sequence). Other sigmas bind to different promoter consensus sequences for genes with specialized functions
a2 (alpha) subunit
Essential for enzyme assembly and involved with activator interactions
B, B’ (beta, beta prime) subunits
Form catalytic core
w (omega) subunit
Provides structural stability
Transcription initiation
RNA pol is DNA template-dependent but primer-independent. Uses ribonucleotide triphosphates (NTPs) as substrate. Holoenzyme binds promoter (closed complex). Unwinding of 12-15 bp DNA into transcription bubble (open complex). Holoenzyme synthesizes first 10 nt (1 nt/sec)
Transcription elongation
Sigma dissociates and is replaced by NusA - allowing RNA pol to complete promoter clearance. Elongation rate of 50-90 nt/sec but can be slowed by secondary RNA structure formation in transcript. Transcription bubble is 12-15 bp and DNA-RNA hybrid is 8 bp. Topoisomerases relieve supercoiling.
Termination results in RNA release and dissociation of core enzyme from DNA.
Rho-dependent termination
Requires Rho (p) protein. Rho is hexameric helicase that binds to rut site on nascent RNA. Translocates 5’-3’. When RNA pol pauses due to RNA secondary structure, termination occurs when/if Rho contacts RNA pol
Rho-independent termination
Requires termination signal in nascent RNA. Occurs due to formation of stable hairpin followed by series of Us. Hairpin causes only weak AU bp between RNA and DNA. DNA-RNA hybrid is unstable and causes RNA disassociation
