Los investigadores utilizan la cristalografía de rayos X para determinar la estructura tridimensional de macromoléculas como los ácidos nucleicos y las proteínas. En esta figura examinaremos el modo en que los investigadores de la Universidad de California, en Riverside, determinaron la estructura de la proteína ribonucleasa, una enzima cuya función implica la unión a una molécula de ácido nucleico. Los investigadores dirigen un haz de rayos X a través de la proteína cristalizada. Los átomos del cristal difractan (desvían) los rayos X en una disposición ordenada. Los rayos X difractados son expuestos a una placa fotográfica y se produce un patrón de puntos conocido como patrón de difracción de rayos X.
Photographic film Diffracted X-rays X-ray source
X-ray beam
Crystal
X-ray diffraction pattern
Utilizando información a partir de los patrones de difracción de rayos X, al igual que la secuencia de aminoácidos determinada por métodos químicos, los científicos construyen un modelo computarizado tridimensional (3D) de la proteína, como este modelo de la proteína ribonucleasa (violeta) unido a una cadena corta de ácido nucleico (verde)
Nucleic acid
X-ray diffraction pattern
3D computer model
Protein
DNA
Synthesis of mRNA in the nucleus mRNA
NUCLEUS CYTOPLASM
mRNA Movement of mRNA into cytoplasm via nuclear pore
Ribosome
Synthesis of protein
Polypeptide
Amino acids
5′ end
Nucleoside Nitrogenous base
Phosphate group Nucleotide 3′ end
Polynucleotide, or nucleic acid
Pentose sugar
Nitrogenous bases Pyrimidines
Cytosine C
Thymine (in DNA) Uracil (in RNA) U T Purines
Adenine A
Guanine G
Pentose sugars
Deoxyribose (in DNA) Nucleoside components
Ribose (in RNA)
5′ end
3′ end Sugar-phosphate backbone Base pair (joined by hydrogen bonding) Old strands Nucleotide about to be added to a new strand
5′ end
New strands
5′ end
3′ end 5′ end
3′ end
Sugar–phosphate backbone
Nitrogenous bases
5′ end
Thymine (T)
Adenine (A)
Cytosine (C)
Phosphate Sugar (deoxyribose) 3′ end
DNA nucleotide
Guanine (G)
Rosalind Franklin
Franklin’s X-ray diffraction photograph of DNA
1 nm 3.4 nm
0.34 nm Key features of DNA structure
5′ end Hydrogen bond
3′ end
3′ end 5′ end Partial chemical structure
Space-filling model
5′ end Hydrogen bond
3′ end
1 nm 3.4 nm
3′ end 0.34 nm Key features of DNA structure
5′ end Partial chemical structure
Space-filling model
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width consistent with X-ray data
Sugar
Adenine (A)
Sugar Thymine (T)
Sugar Sugar
Guanine (G)
Cytosine (C)
Sugar
Sugar Adenine (A)
Thymine (T)
Sugar
Sugar
Guanine (G)
Cytosine (C)
2 nm DNA double helix Histones
Histone tails Histone H1
Linker DNA (“string”)
Nucleosome (“bead”)
Nucleosomes (10-nm fiber)
10 nm
30 nm
Nucleosome 30-nm fiber
Protein scaffold Loops 300 nm Looped domains (300-nm fiber)
Scaffold
700 nm
1,400 nm
Metaphase chromosome
Signal
NUCLEUS Chromatin
DNA
Gene available for transcription Gene Transcription
RNA
Exon Primary transcript Intro RNA processing Tail
Cap
mRNA in nucleus Transport to cytoplasm
CYTOPLASM mRNA in cytoplasm Degradation of mRNA
Translation
Polypeptide Cleavage Chemical modification Transport to cellular destination Active protein Degradation of protein Degraded protein
Histone tails
DNA double helix
Amino acids available for chemical modification
Histone tails protrude outward from a nucleosome
Unacetylated histones
Acetylated histones
Acetylation of histone tails promotes loose chromatin structure that permits transcription
Enhancer (distal control elements)
Proximal control elements Exon
Intron
Exon
Poly-A signal Termination sequence region Intron Exon
DNA Upstream
Promoter Primary RNA transcript 5′ (pre-mRNA)
Transcription
Exon
Intron
Intron RNA
Downstream
Poly-A signal Exon Intron Exon Cleaved 3′ end of primary transcript RNA processing: Cap and tail added; introns excised and exons spliced together
Coding segment mRNA
3′ 5′ Cap
5′ UTR (untranslated region)
Start codon
Stop codon
Poly-A 3′ UTR (untranslated tail region)
Distal control element
Activators
Promoter Gene
DNA TATA box
Enhancer
General transcription factors DNA-bending protein Group of mediator proteins
RNA polymerase II
RNA polymerase II
Transcription Initiation complex
RNA synthesis
Liver cell nucleus
Available activators Enhancer
Control elements
Lens cell nucleus
Available activators
Promoter
Albumin gene
Crystallin gene
Albumin gene not expressed
Albumin gene expressed
Crystallin gene not expressed Liver cell
Crystallin gene expressed Lens cell
Exons
DNA
Primary RNA transcript RNA splicing mRNA
or
Protein complex
Degradation of mRNA
Dicer OR miRNA Target mRNA
Hydrogen bond
Blockage of translation
Proteasome and ubiquitin to be recycled
Ubiquitin Proteasome
Protein to be degraded
Ubiquitinated protein Protein entering a proteasome
Protein fragments (peptides)
Proto-oncogene DNA
Translocation or transposition: gene moved to new locus, under new controls
Gene amplification: multiple copies of the gene
New promoter
Normal growth-stimulating protein in excess
Point mutation within a control element
Oncogene
Normal growth-stimulating protein in excess
Normal growth-stimulating protein in excess
Point mutation within the gene
Oncogene
Hyperactive or degradationresistant protein
MUTATION
Growth factor
Hyperactive Ras protein (product of oncogene issues signals on its own.
G protein Cell cycle-stimulating pathway
Receptor
Protein kinases (phosphorylation cascade)
NUCLEUS Transcription factor (activator)
DNA Gene expression
Protein that stimulates the cell cycle