| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
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Policy | DNASYSTEM
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Readings: Brown, 7:156-159; 8:178-179;
8:181-182; 9:196-208
Outline:
Information in DNA used by the cell is expressed as an RNA: transcription
That RNA whose information is used in Protein Synthesis is messenger RNA (mRNA), whereas an RNA that is used per se is a structural RNA, the most common examples of which are transfer RNA (tRNA) and ribosomal RNA (rRNA) molecules, both of which are used in Protein Synthesis. Small nuclear RNA (snRNA) species are additional structural RNA species, used in eukaryotic cells for splicing out of introns from pre-mRNA molecules. Other small structural RNA species are also used in eukaryotic cells. See Brown, Section 9.1
RNA used in protein synthesis (mRNA) is also called coding RNA, and all other RNA species which are used per se are also called noncoding RNA.
In prokaryotes, transcription is directly coupled to translation or protein biosynthesis. Thus, an mRNA molecule is used for protein biosynthesis often before its synthesis is completed... [Brown, Fig 9.5]
In eukaryotes, mRNA is synthesized in the cell
nucleus and is then transported from the nucleus to sites of protein
biosynthesis in the cytoplasms ... [Brown, Fig. 9.4]
Thus, transcription and translation are not coupled in eukaryotes.
A. Basic process: Initiation,
Elongation, Termination
... [Brown, Fig
8.1]
Initiation occurs at specific DNA sites called Promoters [Brown, Fig 8.8]
Termination occurs at specific DNA sites called Terminators (or transcription termination sites)
Elongation occurs on DNA between Promoters and
Terminators
Example for Trp operon: [Brown, Fig 9.11] and Lac operon
[Brown, Fig 8.8]
Terminology:
Upstream of a Promoter: AWAY from Gene; opposite direction
from transcription
Downstream of a Promoter: INTO the Gene, same direction
as transcription
B. RNA polymerase ... Sigma Factors:
catalyzes RNA synthesis in Transcription ... Transcriptase
In bacteria, there is a single RNA polymerase
E. coli RNA polymerase: Core Enzyme: (alpha)2(beta)(beta-prime)
HoloEnzyme: (sigma)(alpha)2(beta)(beta-prime) ... [Brown, page 179]
Sigma is the Sigma Factor ... MW = 70 kD; hence: (sigma)70
Some Phage encode their own RNA polymerase, eg phage T7 ... simpler enzymes
The difference between Phage and Bacterial RNA polymerases focuses on the key Initiation question:
How does RNA polymerase find Promoters on DNA?
Promoters are DNA sites upstream of gene(s) to be transcribed (expressed) to which HoloRNApol binds and at which the Initiation Process of transcription begins.
Bacterial promoters are many in number and Control of their usage is the main mechanism whereby Bacteria adapt to different environments.
Phage promoters are few in number and are used at specific times in the viral life cycle
HoloRNAPol subunit roles:
Sigma - 70 kD - recognizes the Promoter sequence: promoter binding of Holo enzyme ... [Brown, Fig 8.14]
Beta - ~ 155000 kD - nucleotide binding
Beta-prime - ~ 160000 kD - template binding
Alpha - ~ 40000 - enzyme assembly; binding to rRNA promoters
C. Initiation Process ...
Complexes ... Promoter DNA:
... [Brown, Fig
8.10, 8.11]
The stage at which Control is exerted: when and in what environments will a given gene (or set of genes) be expressed ...
Control factors: Repressor and Activator molecules, mainly Proteins
Finding Promoters is controlled by s70 molecules:
~ 7000 core RNA polymerase molecules per cell, ~ 2000 (sigma)70 molecules
Core RNA polymerase readily binds ANY DNA sequence: loose complex (site)
HoloRNAPol binds 1000x less well to any DNA sequence, falls off quickly ... BUT binds 10000x better to Promoter regions than does CoreRNAPol
Binding of HoloRNAPol to Promoter region: Tight Binary Complex
Binary: two components ... here: HoloRNAPol and DNA
Hence: Sigma Factor the key to RNA Polymerase finding Promoters:
~ 1000 Core Enzymes ... all in Loose Complexes on any DNA sequence
~ 1000 HoloEnzymes in Loose Complexes ... but fall off quickly
~ 1000 HoloEnzymes in Tight Complexes on Promoters
~ 3000 Core Enzymes in Elongation
stage of Transcription
Model for HoloEnzyme finding Promoters in Bacterial Nucleoid:
HoloEnzyme hops on DNA ... if not a Promoter, hops off ... and hops on DNA at new site ... continues this until a Promoter is found
Energy required for moving down the DNA ... no evidence for this
Steps in Initiation once a Tight Complex is formed:
HoloRNAPol binds tightly to Promoter: Tight binary complex
Sigma recognizes Promoter sequences; enzyme covers ~75 bp of DNA
HoloRNAPol catalyzes DNA strand opening: Open binary complex
Only about 9 bp of DNA are unwound ...
HoloRNAPol repeatedly synthesizes
2-9 bases of RNA, which are released:
Abortive Initiation
continues until more than 9 bases are synthesized; Ternary complex formed
Ternary: three components ... here: HoloRNAPol - DNA - RNA
Sigma factor then is released
(Promoter Clearing Time), and Initiation ends
Structure of Promoter: ... [Brown, Page 179, Fig 8.8, Table 8.2]
1. Purine at +1
2. TATAAT (TATA box) at -10;
3. Spacer Region (16-19 bases);
4. TTGACA at -35
Evidence that this is the Important Structure:
Consensus Sequence: compare DNA sequences of many promoters ... [Brown, Table 8.2]
Promoter Mutations:
Up: stronger Promoter;
Down: weaker promoter
Mutational changes fit well with Consensus Sequence, and are mainly in -10 and -35 regions
Evidence for DNA Polymerase binding to Promoter:
DNA footprinting - Nucleotides
involved in Protein Binding:
... [Brown, Section 7.3.1-7.3.2]
sequencing gel analysis of fragments generated by DNase I digestion with and without Protein bound: what backbone bonds are protected ?
Similarly: chemically modify DNA bases, e.g. Dimethyl Sulfate (DMS) to modify Guanines (one of the Maxam-Gilbert DNA sequencing chemistries)
1. With and without Protein bound: which bases are protected from modification?
2. Modify bases and then bind Protein: which modified bases prevent binding?
Results: important bases in Promoters are
1) in mainly -10 and -35 regions
2) are on ONE SIDE of the DNA duplex => HoloRNAPol binds to one DNA side
Simpler Approach to show Binding: Gel Retardation
When a protein binds a DNA fragment, the migration rate of the DNA fragment in an Agarose Gel is slowed down or "retarded" ... [Brown, Fig 7.13]
This provides a "qualitative" assay, to show that a protein has bound the DNA fragment in question
D. Elongation Process ...
Reaction:
... [Brown, Section
9.2; Research Brief 9.1]
catalyzed by CoreRNAPol ... about 100 nucs/sec (vs 105 for Replication)
Similar to DNA synthesis but no Primer is required ... no Exonuclease activity:
DNA + 4 rNTPs --> DNA + RNA + PPi
RNA has ppp at its 5' end ...
[Brown, Fig 9.6]
RNA polymerase structure changes during Elongation:
CoreRNAPol initially 35 bp in length ... 35 x 3.4 = 120 Å ... 3.5 DNA turns
Length shrinks at 9 RNA nucleotides are synthesized ... to about 27 bp = 90 Å
Length springs back to 35 bp
=> Enzyme ratchets down the DNA, ala a slinky
Supercoiling:
Unwinding and movement of RNA pol increases turns ahead of fork, decrease turns behind the fork
W-C twists (T) remains constant ... hence, changes in turns seen as changes in supercoiling
Increase in turns ahead of fork: Increase in supercoils, negative supercoils decrease ... DNA gyrase introduces more negative supercoils
Decrease in turns behind fork: Decrease in supercoils, negative supercoils increase ... Topo I relaxes these excess negative supercoils
(Topo I yeast mutant: negative supercoil density increases during transcription)
E. Termination Process ... Rho-dep and Rho-indep:
Termination of transcription occurs when CoreRNAPol encounters a Terminator or Termination Site (t)
Termination sites in bacteria
are of 2 types:
Rho-independent Termination Sites: ... [Brown, Fig 9.8 A]
These are characterized by the RNA structure and involve no known Protein or other factors. The structure is a G,C-rich Stem-Loop ending in a run of U's
The Stem-Loop is thought to slow down CoreRNAPol, and the weak basepairing in the U region of the RNA to the DNA template releases the RNA. CoreRNAPol is then released, to again hop onto DNA (loose binding complex)
Rho-dependent Termination Sites: ... [Brown, Fig 9.8 B]
The less common of the two types of sites, these sites require the Protein factor called Rho, isolated via its property to terminate otherwise very long RNA
Rho sites are characterized only by high-C, low-G regions
Rho can bind RNA and, using ATP as energy, can move along the RNA. It is thought to terminate by "catching" CoreRNAPol which has paused at a Rho termination site, and then catalyzes unwinding and release of the RNA from the DNA.
Much, however, is not yet understood here, eg why only Rho sites if only pausing of CoreRNAPol is needed?
F. Transcription Control: Different Sigma Factors; Anti-Termination
Control of Gene Expression occurs mainly at Initiation of the Transcription Process.
In prokaryotes, this control is of two types:
1. Adaptation to Environment
Changes:
This type of control needs to be of an "on or off",
reversible type. Prokaryotes such as bacteria need to be able
to respond or "adapt" to an environmental change, e.g.
new sugar source or loss of an amino acid, and then need
to respond or "adapt" back to the original environment
as well.
The primary control mechanism for this is the Repressor mechanism characteristic of Operon Theory (see Lecture 18)
Other mechanisms also exist,
including those usually found for the second type of control.
For example, operon specific Sigma factors ... examples:
1) Heat shock 32-kD sigma called
Sigma-32.
Upon shift to an elevated temperature, bacteria show the Heat
Shock Response in which several operons of genes (in total,
a regulon) are "turned on" or expressed via CoreRNApol
plus Sigma-32 recognizing Promoters containing a -10 and
-35 sequences recognized by the Sigma-32 initiation recognition
factor. ... [Brown, Fig 8.14]
2) SOS response to UV irradiation
This is a combination of Operon Theory via the LexA repressor
... and the unique mechanism of UV-induced DNA damage activation
of the RecA repressor, which activation leads to inactivation
of the LexA repressor (and a few others).
2. Temporal Control - Expression
of Genes as a Function of Time:
This type of control is a "one way street", irreversible
type: genes are turned on or turned off as a function of time
after an event.... and often these events are irreversible.
In eukaryotes, developmental processes, coupled with cell differentiation, are examples of irreversible temporal control events.
The temporal events which occur following viral infection of eukaryotic host cells, or phage infection of prokaryotic host cells, constitute another general example of Temporal Control. Several types of molecular mechanisms exist following phage infection of bacteria that result in a temporal control of expression of viral genes as a function of time after infection, including:
1) Induction of new Sigma
Factors.
For example, a new Sigma Factor can be expressed as an "Early
Gene", which then together with CoreRNApol results in expression
of "Middle Genes". One of these Middle Genes can encode
yet another Sigma Factor, which then leads to expression of "Late
Genes". Example: E. coli phage SP01.
2) Modification of Core
RNA Polymerase subunits.
For example, a protein expressed as an "Early Gene"
can modify one of the Core RNA Polymerase subunits, e.g. via methylation,
phosphorylation, or acetylation. The modified Core RNA Polymerase,
together with the host Sigma Factor, is then specific for expression
of "Middle Genes". Example: E. coli phage T4
3) Expression of a new Phage-specific
RNA Polymerase:
For example, a new RNA Polymerase can be expressed as an "Early
Gene", which then results in expression of "Middle Genes"
from new Promoters. Example: E. coli phage T7
4) Anti-termination:
For example, a protein expressed as an "Early Gene"
can interact with RNA polymerase to prevent termination
at a transcription terminator used for Early Gene expression.
This results in expression of additional genes, and these are
the "Middle Genes". Note that in this mechanism, the
Early Genes are also expressed as Middle Genes, and that the same
Promoters are used for both Early and Middle Gene expression ...
[Brown, Fig 9.9]. Example: E. coli phage Lambda. Lambda
N gene, as an "Early-Early" or "Immediate-Early"
or "Early" gene product functions as an Anti-terminator
at two phage Lambda Rho-dependent Terminators, resulting in expression
of "Early" or "Delayed-Early" or "Middle"
lambda genes ... [Brown, Box 9.1].
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith