Molecular Biology in Medicine 医学分子生物学

Molecular Biology in Medicine

医学分子生物学许正平

[email protected]

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)

Genetic Information Transfer

遗传信息的传递 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 Regulation of Gene Expression

基因表达的调控 ( 包括 miRNA 、 RNAi)

分子生物学主要内容

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 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

基因操纵技术： Gene knock-out/knock-in

RNA interference (RNAi)

分子生物学主要研究技术蛋白质相互作用：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)

蛋白质鉴定：质谱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。

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

医学分子生物学

教材：医学分子生物学（第 3 版）

参考书： Molecular Cell Biology Gene

More Information: Literature Internet

I. Introduction

The Central Dogma

I. Introduction

How many genes in the human genome?Gene Expression

I. Introduction

FACT 1: an uniform genome in almost every cell of an organism

FACT 3: the shape and function of each type of cell are different

FACT 2: the proteome in each type of cell is different

I. Introduction

the actions and properties of each cell type are determined by the proteins it contains

FACT 1: an uniform genome in almost every cell of an organism

transcription of different genes largely determines the actions and properties of cells

FACT 3: the shape and function of each type of cell are different

FACT 2: the proteome in each type of cell is different

I. Introduction

the types and amounts of the various proteins in a cell

the concentration of mRNA and the frequency at which the mRNA is translated

which genes are transcribed and their rateof transcription in a particular cell type

TRUTH: the gene is differentially expressed

regulation

same genome in all cells of an organism

regulation

regulation

I. Introduction

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

II. Transcription

DNA (gene)

RNA polymerase

Regulatory Proteins

Key Players

promoter

A

startpoint terminator

Transcription Unit

template

upstream downstream

enhancer

II. Transcription

Primary transcript is the original unmodified RNA product correspondingto 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.

Key Terms

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

II. Transcription

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

II. Transcription

RNA Polymerase

The promoters for RNA polymerases I and II are (mostly) upstreamof 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 setof 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.

Promoter

II. Transcription

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 constitutivelyspecific: usage is regulated; define a particular class of genes

These sites are organized in different combinations

Cis-acting Element

II. Transcription

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.

Enhancer

two characteristics:1. the position of the enhancer need not be

fixed.2. it can function in either orientation.

II. Transcription

promoter enhancer

position fixed variable

action direction one way either orientation

the density of regulatory elements sparse Heavy (closed packed)

redundancy in function no yes

cooperativity between the binding of factors

sequential great

The Difference between Promoter and Enhancer

The distinction between promoters and enhancers is operational, rather than imply a fundamental difference in mechanism

II. Transcription

Most Eukaryotic Genes Are Regulated by Multiple Transcription-Control Elements

(a) Genes of multicellular organisms contain both promoter-proximal elements and enhancersas 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.

II. Transcription

Fact: Regulatory elements in eukaryotic DNA often are many kilobases from start sites

Finding Regulatory Element in Eukaryotic DNA

II. Transcription

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.

II. Transcription

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

Another name: accessory factor

II. Transcription

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

Accessory Factors

II. Transcription

II. Transcription

Fou

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tage

s in

T

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on

1. On the genome Which gene(s) to be transcribed? Basic events: Protein binding and/or modification 2. On a specific gene If the gene can be transcribed successfully?

3. On a transcript If the transcript could be correctly spliced? If the transcript could be correctly edited?

Regulation Levels

Key determinant: Cell Signaling!

III. Regulation of transcription

Potential regulation points

Activation of gene structure↓

Initiation of transcription↓

Processing the transcript↓

Termination of transcription↓

Transport to cytoplasm

the overwhelming majority of regulatory events occur at the initiation of transcription


5 potential control points:

“Active” Structure

Major Control Point

Alternative Splicing

Regulatory Proteins

the overwhelming majority of regulatory events occur at the initiation of transcription

Key player: regulatory transcription factors

Two questions:1. How does the transcription factor identify its group of target genes?2. How is the activity of the transcription factor itself regulated in response to intrinsic or extrinsic signals?


Answer to question 1

The genes share common response element

Structure feature: contain short consensus sequence

Examples:

HSE: heat shock response element; recognized by HSTF

GRE: glucocorticoid response element

SRE: serum response element

MRE: metal response element


Regulatory region in MT gene

BLE: basal level element; TRE: TPA response element

General Principle: any one of several different elements, located in either anenhancer or promoter, can independently activate the gene.


? = MTF-1

Answer to question 2

Signal transduction

Key events:

1. Protein synthesis

2. Protein modification

3. Ligand binding

4. Protein cleavage

5. Inhibitor release

6. Mutation


The activity of a regulatory transcription factor may be controlled by synthesis of protein, covalent modificationof protein, ligand binding, or binding of inhibitors that sequester the protein or affectits ability to bind to DNA.

Reg

ula

tion

Mod

es o

f T

ran

scri

pti

on

Fac

tor

mutations of the transcriptionfactors give rise to factors thatinappropriately activate, or prevent activation, of

transcription


Eukaryotic transcriptional control operates at three levelsduring the stage of initiation

1. changes in chromatin structure directed by activators and repressors

2. modulation of the levels of activators and repressors(gene expression)

3. change the activities of activators and repressors


Gene differential expression

IV. RNA Processing

INTRODUCTION

Facts:

1. Genes are interrupted, and mRNAs are uninterrupted

2. The primary transcript has the same organization as the gene

3. Most mRNAs have 5’ cap and 3’ poly(A) tail

4. Heterogeneous nuclear RNAs (hnRNA) exist in the nucleus

5. RNA contains rare bases

Mechanism:

RNA splicing: remove intron

RNA modification: 5’ capping, 3’ polyadenylation, base modification

INTRODUCTION

The initial primary transcript synthesized by RNA polymerase IIundergoes several processing steps before a functional mRNA is produced:

5’ capping 3’ cleavage/polyadenylation RNA splicing

RNA splicing is the process of excising the sequences in RNA that correspond to introns, so that the sequences corresponding to exonsare connected into a continuous mRNA.

IV. RNA Processing

Overview of mRNA Processing in Eukaryotes

The poly(A) tail: ~250 A in mammals, ~150 in insects, ~100 in yeasts. For short primary transcripts with few introns, polyadenylation, cleavage, and splicing usually follows termination. For large genes with multiple introns, introns often are spliced out of the nascentRNA before transcription of the gene is complete.

IV. RNA Processing

Th

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osom

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cle

The splicing snRNPs associate with the pre-mRNA and with eachother in an ordered sequence to form spliceosome

ATP is needed to provide theenergy necessary for rearrangements of the spliceosome structure

IV. RNA Processing

Alternative splicing

Mechanisms:• use of different startpoints or termination sequences• a single primary transcript is spliced in more than one

way, and internal exons are substituted, added, or deleted

Definition: a single gene gives rise to more than one mRNA sequence

Key:what controls the use of such alternative pathways?Proteins? ncRNA?

IV. RNA Processing

Alternative splicing

Mechanisms:• use of different startpoints or termination sequences• a single primary transcript is spliced in more than one

way, and internal exons are substituted, added, or deleted

Definition: a single gene gives rise to more than one mRNA sequence

Key:what controls the use of such alternative pathways?Protein(s)!

IV. RNA Processing

The Troponin （肌钙蛋白） T (muscle protein) pre-mRNA is alternatively spliced to give rise to

64 different isoforms of the protein

Constitutively spliced exons (exons 1-3, 9-15, and 18)

Mutually exclusive exons (exons 16 and 17)

Alternatively spliced exons (exons 4-8)

Exons 4-8 are spliced in every possible waygiving rise to 32 different possibilities

Exons 16 and 17, which are mutually exclusive,double the possibilities; hence 64 isoforms

IV. RNA Processing

Trans-(intermolecular) splicing

Splicing is usually cis-reaction (intramolecular), but trans-(intermolecular) splicing have been found (very rare). These reactions probably occur by splicesome formation with the appropriate site sequences on each molecule.

trypanosomes and euglenoids: all the mRNAsCaenorhabditis elegans: 10-15% of the mRNAsHuman?

IV. RNA Processing

Initiation of Protein Synthesis

V. Initiation of Protein Synthesis

Critical event:begin protein synthesis at the start codon, thereby setting the stagefor the correct in-frame translation of the entire mRNA.

Main mechanisms:Base pairing between mRNA and rRNABase pairing between mRNA and tRNAMet-tRNAi

Met can only bind at the P site to begin synthesis

Participants: Met-tRNAi

Met

mRNA IFs small subunit large subunit

Protein translation

Two types of methionine tRNA are found in all cells

same aminoacyl-tRNA synthetase (MetRS) charges both tRNAs with methionine


Eukaryotic initiation of protein synthesis


Model of protein synthesis on circular polysomes and recycling of ribosomal subunits

PABI and eIF4 (4G and 4E) can interact on mRNA to circularize the molecule


The nascent polypeptide chain must undergo folding and, in many cases, chemical modification and cleavage to generate the final protein

Folding:Theoretically: any polypeptide chain containing n residues could, in principle, fold into 8n conformations. Fact: adopt a single conformation (native state)

a single, energetically favorable conformation Mechanism: the amino acid sequence provides the information for protein folding

Protein Maturation

Modification: N terminal C terminal Certain sites btw N and C terminus

VI. Protein Processing

Nearly every protein in a cell is chemically altered after its synthesis in a ribosome, thus alter its activity, life span, or cellular location of proteins,

depending on the nature of the alteration.

Two categories:chemical modification involves the linkage of a chemical group to the terminal amino or carboxyl groups or to reactive groups in the side chains of internal residues may be reversibleProcessing involves the removal of peptide segments and generally is irreversible

Protein Alteration


The internal residues in proteins can be modified by attachment of a variety ofchemical groups to their side chains:phosphorylation (Ser, Thr, Tyr) glycosylation (Asp, Ser, Thr)ubiquitinationothers

Examples of modified internal residuesproduced by hydroxylation, methylation, and carboxylation

Protein Modification


Protein Cleavage

most common form:

residues are removed from the C- or N-terminus of a polypeptide

by cleavage of the peptide bond in a reaction catalyzed by

proteases.

Proteolytic cleavage is a common mechanism of activation or

inactivation

Proteolysis also generates active peptide hormones

EGF; insulin


Protein Degradation

Two Pathways

extracellular: digestive proteases

intracellular

lysosomes

cytosolic mechanisms

The ubiquitin-mediated pathway is the best-understood cytosolic pathway.In ubiquitinating enzyme complex, different conjugating enzymes recognize different degradation signals in target proteins.

ubiquitin-conjugating enzyme E1: Arg-X-X-Leu-Gly-X-Ile-Gly-Asxcertain residues at the N-terminus favor rapid ubiquitination


基因突变、多态性：个体易感性、疾病发生表观遗传修饰：功能改变蛋白质量 / 结构 / 构象 / 功能改变：非正常功能、疾病调控异常：非正常功能、疾病

分子生物学与医学

医学分子生物学的应用

阐明生理 / 病理现象的分子机制遗传病诊断（基因诊断）发现疾病的生物标志物，为诊断服务疾病的生物治疗药物研发与评价（药物基因组学）个性化医疗

Thank you for your attention!

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Molecular Biology in Medicine 医学分子生物学