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DNA
Transcription
RNA
Translation
Protein
Phenotype
The assumptions of the central dogma are sound, but there are exceptions!
Ribozymes
FUNCTION
Interactions
FUNCTION
Ribozymes favor the concept that a more primitive RNA-based ancestral form of life may have preceded the DNA-, RNA-, protein-based life as we know it now.
Transcription
The first step of the expression of a gene (DNA) is to transcribe the information contained in the nucleotide sequence from one strand (template or antisense strand) into an RNA. The product of this transaction is a transcript. If a gene carries information for the assembly of a protein, the transcript is a messenger RNA (mRNA).
Translation
The information in mRNAs is decoded by ribosomes and translated by alignment of three-nucleotide codons with corresponding tRNAs that deliver the corresponding amino acids.
The messenger: a general view of transcription and Coding strand of DNA translation
Template strand of DNA
mRNA
RNA precursors
Base
O
Ribonucleoside triphosphate
RNA Structure
Primary structure: order of the nucleotides read 5 3 5 ACUCAUCGGCACGUCAUGCUGAUAUCCGGCUUGACACU 3 Secondary structure: regions of base pairing (double-stranded helical regions) Tertiary structure: folding of the entire RNA chain
Only non-covalent hydrogen bonding is involved in secondary and tertiary structure formation
Stem-loop
Fig 2.2
Pseudoknot
5 3
Messenger contains information for mRNA assembly of amino-acid sequence of protein by ribosomes and tRNA tRNA Transfer codon recognition in partnership with ribosome and transfer of amino acid into polypeptide Ribosomal component of ribosome, binding of mRNA, tRNA and formation of peptide bond between amino acids Diverse roles: primers, gene regulation, guide RNAs, RNA processing and degradation, cofactor in protein complexes
rRNA
Small RNAs
RNAs and DNAs can be separated by size in sucrose gradients by centrifugation and by gel-electrophoresis
Separation by Electrophoresis
5S
5 3
Direction of reading of template DNA strand:
3 5
Do you remember the principles of nucleic-acid synthesis? Ch. 1!
Size aa Da
329 1342 1407 91 613 36,511 150,616 155,159 10,237 70,263
Gene Function
rpoA rpoB rpoC rpoZ rpoD
Assembly of RNAP, binding of some regulatory proteins, catalysis. Catalysis of chain initiation and elongation. Binding to DNA template. Restores denatured enzyme to fully functional form. Directs enzyme to corresponding promoters.
DNA
Fig 2.6
pincer
Holoenzyme
RNA synthesis proceeds for ~8 9 nt along the template, is released. Transcription continues until the polymerase with its transcription bubble reaches a transcription termination (t ) site. The RNA and the polymerase dissociate and are released from the DNA.
Fig. 2.7
Transcription Cycle
binding
Termination Closed complex (RPC) Transcription elongation complex (TEC) release and elongation Open complex (RPO)
Promoter recognition
DNA isomerization
Escape
Initiation
Abort
UP element Extended 10
Down arrows indicate contact points with core RNAP proteins Electrostatic interactions Direct hydrogen bonds Indirect hydrogen bonds potential hydrophobic (van der Waals) contacts.
From S. Borukhov and E. Nudler 2003. Curr. Opinion. Microbiol. 6: 93-100
Coding-strand
Template-strand
The RNA polymerase adds from 30 to 100 nucleotides per second to the growing RNA molecule (6000 maximum per min).
Each type of factor recognizes its own unique class of promoter sequences
See next page
Gene
rpoD rpoN (ntrA, glnF) rpoH rpoS rpoE rpoF fecI
Function
Principal sigma factor (housekeeping gene transcription). Nitrogen-regulated gene transcription. Heat-shock gene transcription. Gene expression in stationary phase cells. Periplasmic stress response proteins. Expression of flagellar operons. Regulates the fec genes for iron dicitrate transport.
Transcription Termination
There are two kinds of transcription termination: 1. Factor independent termination and 2. Factor-dependent termination.
Factor-independent transcription termination occurs at sites in the DNA that include a region of two-fold symmetry followed by a stretch of at least four As in the template strand.
RNA
DNA
Any transcription-pause site in DNA DNA Polysomes In prokaryotes, translation is coupled to transcription.
IMPORTANT: Factor-dependent transcription occurs preferentially when the translation of a nascent mRNA is stalled.
binds
rut
Stalled ribosome
Fig 2.19
Rifampin is a member of the rifamycins, macrocyclic lactone antibiotics that inhibit transcription at the initiation stage, but do not block elongation once initiation is complete. Rifamycins bind to the -subunit in the wall of the active-site channel of the RNAP of bacteria, mitochondria and chloroplasts. Two or three nucleotides are polymerized. MORE
Rifampin
Action of Rifampin
pppApN and pppGpN are the most common products. The Streptovaricins are related compounds that have the same action as rifampin, except that they also can block transcript elongation.
Binds to the major groove of DNA in G/C rich regions. Inhibits transcription and replication
Actinomycin D
RNA Editing
Processed RNA products Further processing and modification of bases (maturation) MATURE rRNAs and tRNAs
Fig. 2.20
RNase
B. subtilis
tRNA processing
Rnase P consists of an RNA dimer (same sequence, catalytic subunit) complexed with a dimer of a small protein. It is a ribozyme.
RNA modification
Structure of a mature tRNA
Dihydrouridine
IV
III
II
Fig. 2.21
Degradation of mRNA
Some of the enzymes involved also participate in rRNA and tRNA processing
Box 2.5
1. ~1500 nucleotids 2. Many modified bases 3. Compact 3-D folding 4. Complexed with 21 proteins 5. Highly conserved
16S rRNA
RNA processing
Introns and splicing
Splicing: Removal of parasitic DNA information from RNA
Group II introns
Group I introns
Box 2.6
N-extein
Intein
C- extein
284
454
738
1071
Box 2.6
* deleted U u added U
uGAUACAAAAAAACAUGACUACAUGAUAAGUAuCAuuuuAuGuuAuuuuuGGuAGuuuuuuuACAuu uGuAuCGuuuuACAuuuG*GUCCACAGCAuCCCG***CAGCACAuG**GuGuuuuAuGuuGuuuAuuGuA uuuuuGuGGuGA*AuuuAuuGuuuA**UAUUGAuUGuAuuAuA***G*GuuAUUUGCAUCGUGGUACAG AAAAGUUAUGUGAAUAUAAAAGUGUAGAACAAUGUCUUCCGuAUUUCGACAGGUUAGAuuAuG uuA*GuGuuuGuuGuAAuGAGCAuuuGuuGuCuuuA***UGuuuuGAGuAuAuGuuGCGAuGuuGuuuGu CGuuACGuuGuGCAuuuAuGCGuuuAuuAAuuGuA****GAAuuuAC***CCGuAGuuuuAAuGGuuuGuu GuGuAuAuCAuGuAuGGuuuuGG*AuuuAGGuuGuuuGuCUCCGuuG*UUAuGAuCAuuuGAGGAA*** CG*UGACAAAuuGAuGACAuuuuuuGAuuuAuG**UUGuGGuuGuCGuAuGCAuuuGGCUUUCAuGGu uuuAuuA*GGuAUUCUUGAUGAuuuuGuuuuuGGuuuuGuuGAuuuuuuGuuGuuGuuGA***UAAuAuC AuGuuuGuuuGuuAuGGAuuGuuAuGAuuuGuuAuuuGuGGGuAAUCGuuuAuuuUAuuuGCGuuuGC** *GuGGuuuGuCAuuuuuuGAuuuAuAuGAuuuA**GuuuuuA**A**UAGuuuAAGuGGuGuuuuGuCuCGu uCGuuAGGuAuGGuGuGAGAuuGUCGuuuAuuuAGuuGuuA****UGA*****GuUGuAuuuuAuGuuuuG uuAuGAuuAuuGuuuuuGuuuuAuAGGuGAuGCAuuuGA*UCGuuuAuuuuuACGuuuGuuuGAUAuGC GuAuGAGuuuGuuGAuuuGuAAGCAAuGuuuuuuuGuuGGuuuuuuuGuuuuuG*****GuuuuGuuuGuuu GuuuG**AuuAuuuAuAuuGuGAuAuuACCAuuG****AGACCAuuAuuAuGuuAuuuuAuAGuuuGuGGu GuuGuuGuuuGCCGGGuAuA*UCAuuuGC*UUGUGuuGAACACCCCAAAGGuGA***GuAuuGuuuGu uAuuA****UGuuuuuGuGuuGGuuuAuGuuCUCGuuuACGuuuGCGuuGuGCGGAuuuuuuGCA*UAUU UGuuuAuuGGAuGuuuGuuuGCGuGGuuuuuuAuuGCAuGAuuuAGuuGC***C*GuuuuAGGuAAuAuu GAuGuuGuuuuuGGAuCCGUAGAUCGuuA*GuuuuAuAuGuG**A******GGUUAUUGuAGGAUUGUU UAAAAUUGAAUAAAAA Courtesy of Dr. Donna Koslowsky
DNA
5 ATATAAAAGCGGGAGTTA
EDITOSOME
UU UU Edited segment 5 AUAUAAAAGCGGGAGUUAUUUUUAUUAUUUUUU 3 .....*... .... 3 UUUUUUUUU UAAAAGUAAUAAA 5 C C Tether G Anchor A A A U C U A G A CC A AC GUIDE
Untranslated regions (UTRs) and secondary structure modifications of nuclear RNAs in eukaryotes