Transcript 医学分子生物学

Molecular Biology in Medicine
医学分子生物学
许正平
[email protected]
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2012/popular-chemistryprize2012.pdf
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2012/
popular-chemistryprize2012.pdf
一、导论
医学分子生物学
定义：
从分子水平研究人体在正常和疾病状态下生命活动
及其规律的一门科学
重点：
人体生物大分子和大分子体系的结构、功能、相互
作用及其同疾病发生、发展的关系
分子医学 Molecular Medicine
The greatest intellectual revolution of the
last 40 years may have taken place in biology.
Can anyone be considered educated today
who does not understand a little about
molecular biology?
─F. H. Westheimer (Harvard University)
Molecular Medicine
Molecular medicine is a broad field, where physical,
chemical, biological and medical techniques are
used to describe molecular structures and
mechanisms, identify fundamental molecular and
genetic errors of disease, and to develop molecular
interventions to correct them.
GFP and other FPs (fluorescent proteins)
Telomere DNA protects the chromosomes
Vesicle trafficing: get the right molecules to
the right place at the right time
http://www.nobelprize.org/nobel_prizes/medicine/lau
reates/2013/press.html
分子生物学主要研究内容
 Gene & Genome基因与基因组
 Gene Transcription 基因转录
 RNA Splicing & Editing RNA剪切与加工
 Protein Synthesis & Processing
蛋白质合成与加工
 Protein Structure & Function蛋白质结构与功能
 non-coding RNA非编码RNA
分子生物学主要研究技术
 分离、纯化（主要是生物大分子）
 克隆、表达
 序列分析 （Sanger Seq & Next Generation Seq)
 PCR（多聚酶链式反应 ）
 凝胶电泳：琼脂糖凝胶电泳；SDS－聚丙烯酰胺凝胶电泳
（SDS-PAGE）；等电聚焦电泳；双向电泳
 印迹技术：Southern blotting; Northern blotting;
Western blotting
 微阵列技术：genechip, microarray, protein chip
分子生物学主要研究技术
基因操纵技术：Gene knock-out/knock-in
RNA interference (RNAi)
蛋白质翻译调控：ribosome profiling; GTI-seq
蛋白质鉴定：质谱
蛋白质相互作用：酵母双杂交、免疫共沉淀（Co-IP）、
pull-down、FRET、表面等离子共振技术(SPR)
蛋白质与核酸相互作用：ChIP、ChIP-on-chip
 研究生物大分子三维结构常用的实验手段：
X射线晶体学、核磁共振、电子显微学、原子力显微镜
以及X射线小角散射等。
二、基本知识介绍
人类基因与基因组
人类基因典型结构
中心法则 The Central Dogma
人细胞中的基因表达
Some Facts in Human Cells
FACT 1:
an uniform genome in almost every cell of human body
FACT 2:
the proteome in each type of cell is different
FACT 3:
the shape and function of each type of cell are different
TRUTH: the gene is differentially expressed
same genome in all cells of an organism
regulation
which genes are transcribed and their rate
of transcription in a particular cell type
regulation
the concentration of mRNA and the
frequency at which the mRNA is translated
regulation
the types and amounts of the various proteins in a cell
Gene differential expression
Gene Expression Occurs by a Two-Stage Process
Transcription:
generates a single-stranded RNA identical in sequence with
one of the strands of the duplex DNA
Three principal classes of products:
message RNA (mRNA)
transfer RNA (tRNA)
ribosomal RNA (rRNA)
Principle: complementary base pairing
Translation:
converts the nucleotide sequence of an RNA into the sequence
of amino acids comprising a protein
each mRNA contains at least one coding region that is
related to a protein sequence
Gene Transcription
Key Players
DNA (gene)
RNA polymerase
Regulatory Proteins
enhancer
promoter startpoint
template
terminator
A
Transcription Unit
upstream
downstream
Gene Transcription
Key Terms
Primary transcript is the original unmodified RNA product corresponding
to a transcription unit.
Promoter is a region of DNA involved in binding of RNA polymerase
to initiate transcription.
RNA polymerases are enzymes that synthesize RNA using a DNA template
(formally described as DNA-dependent RNA polymerases).
Terminator is a sequence of DNA, represented at the end of the transcript,
that causes RNA polymerase to terminate transcription.
Transcription unit is the distance between sites of initiation and termination
by RNA polymerase; may include more than one gene.
RNA Polymerase
Transcription in eukaryotic cells is divided into three classes.
Each class is transcribed by a different RNA polymerase:
 RNA polymerase I:
 RNA polymerase II:
 RNA polymerase III:
RNA Polymerase
Transcription in eukaryotic cells is divided into three classes.
Each class is transcribed by a different RNA polymerase:
 RNA polymerase I: rRNA; resides in the nucleolus
 RNA polymerase II: mRNA, snRNA; locates in the nucleoplasm
 RNA polymerase III: tRNA and other small RNAs; nucleoplasm
Promoter
The promoters for RNA polymerases I and II are (mostly) upstream
of the startpoint, but some promoters for RNA polymerase III lie
downstream of the startpoint.
Each promoter contains characteristic sets of short conserved
sequences that are recognized by the appropriate class of factors.
RNA polymerases I and III each recognize a relatively restricted set
of promoters, and rely upon a small number of accessory factors.
Promoters utilized by RNA polymerase II show more variation in
sequence, and are modular in design.
Cis-acting Element
Short sequence elements (cis-acting elements): bind by
accessory factors (transcription factors)
The regulatory region might exist in the promoters of certain
eukaryotic genes.
Location: usually upstream and in the vicinity of the startpoint.
These sites usually are spread out over a region of >200 bp.
common: used constitutively
specific: usage is regulated; define a particular class of genes
These sites are organized in different combinations
Enhancer
Enhancer element is a cis-acting sequence that increases the
utilization of (some) eukaryotic promoters.
 The components of an enhancer resemble those of the promoter.
Involve in initiation, but far from startpoint.
 Are targets for tissue-specific or temporal regulation.
 Function in either orientation and in any location (upstream or
downstream) relative to the promoter.
two characteristics:
1. the position of the enhancer need not be
fixed.
2. it can function in either orientation.
The Difference between Promoter and Enhancer
position
action direction
the density of regulatory elements
redundancy in function
cooperativity between the binding
of factors
promoter
enhancer
fixed
variable
one way
either orientation
sparse
Heavy (closed
packed)
no
yes
sequential
great
The distinction between promoters and enhancers is operational,
rather than imply a fundamental difference in mechanism
Most Eukaryotic Genes Are Regulated by
Multiple Transcription-Control Elements
(a) Genes of multicellular organisms contain both promoter-proximal elements and enhancers
as well as a TATA box or other promoter element. Enhancers may be either upstream or
downstream and as far away as 50 kb from the transcription start site. In some cases,
promoter-proximal elements occur downstream from the start site as well.
(b) Most yeast genes contain only one regulatory region, called an upstream activating
sequence (UAS), and a TATA box, which is ≈90 base pairs upstream from the start site.
Finding Regulatory Element in Eukaryotic DNA
Fact: Regulatory elements in eukaryotic DNA
often are many kilobases from start sites
Transcription Factor
Any protein that is needed for the initiation of transcription, but
which is not itself part of RNA polymerase, is defined as a
transcription factor.
binds to DNA (trans-acting factor):
recognize cis-acting elements
interacts with other protein:
recognize RNA pol, or another factor
The common mode of regulation of eukaryotic transcription is positive:
a transcription factor is provided under tissue-specific control to activate
a promoter or set of promoters that contain a common target sequence.
Regulation by specific repression of a target promoter is less common.
Another name: accessory factor
Accessory factors are needed for initiation, principally
responsible for recognizing the promoter.
Interaction with DNA, RNA polymerase, and/or another
factors.
Three groups:
1. General factors
2. Upstream factors
3. Inducible factors
Accessory Factors
 general factors: required for the mechanics of initiating RNA
synthesis at all promoters; form a complex surrounding the
startpoint with RNA pol, and determine the site of initiation.
basal transcription apparatus (pol + GF)
 upstream factors: DNA-binding proteins that recognize specific
short consensus elements located upstream of the startpoint.
not regulated; ubiquitous; act upon any promoter that contains
the appropriate binding site on DNA.
 inducible factors: function in the same general way as the
upstream factors.
have a regulatory role: control transcription patterns in time and
space
Four Stages in Transcription