Molecular Biology Lecture 4

8/12/2019 Molecular Biology Lecture 4

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BIOL321 - Transcription

Cristofre Martin

Department of Biochemistry

St. George’s University



There are two major steps that occur during protein

synthesis:

a) Transcription - the synthesis of a single-stranded RNA copy

of a segment of DNA.

b) Translation - the conversion of the messenger RNA base

sequence information into the amino acid sequence of a protein polypeptide.

The “Central Dogma” of Molecular Biology

DNA RNA ProteinTranscription Translation

Reverse transcription

Replication



Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

The Basic Transcription Process

1. The DNA unwinds in a short region next to a gene.

2. RNA polymerase catalyzes the synthesis of an RNA

molecule in the 5’ to 3’ direction along the 3’ to 5’ template

strand of the DNA.




RNA polymerase

catalyzes thesynthesis of an

RNA molecule in

the 5’-to-3’

direction along the3’-to-5’ template

strand of the DNA.

Ribonucleoside

triphosphates are

the RNA precursor

molecules used for

RNA synthesis.



01_12.jpg

During transcription only one strand of DNA is required as a template.

There is evidence that genes can be transcribed from any strand.

The strand serving as the template for transcription is called the

antisense strand and the other strand is called the sense strand (same

sequences as the RNA)



Comparison of DNA synthesis and RNA synthesis:

RNA DNA

RNA polymerase DNA polymerase

NTPs precursor dNTP precursor

no primer needed primer required for initiation

uracil pairs with adenine thymine pairs with adenine



There are four different types of RNA molecules:

1) mRNA (messenger RNA) encodes the amino acidsequence of a polypeptide. mRNAs are the

transcripts of protein coding genes.

2) tRNA (transfer RNA) brings amino acids to the

ribosome during the translation process.3) rRNA (ribosomal RNA) combines with ribosomal

proteins to form the ribosome. mRNA is translated

into protein at the ribosome.

4) snRNA (small nuclear RNA) combines withcertain proteins and is involved in RNA processing

(example mRNA splicing) in eukaryotes.



In bacteria, there is only a single RNA polymerase:

-composed of the core polymerase plus sigma factor

There are three different RNA polymerases in eukaryotes:

1) RNA polymerase I: 5.8S, 18S, and 28S rRNA genes

2) RNA polymerase II: all protein-coding genes (mRNA) and

some snRNAs

3) RNA polymerase III: tRNA genes and some snRNAs


http://slidepdf.com/reader/full/molecular-biology-lecture-4 9/26Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Regions of a prokaryotic gene:

1) Promoter: located upstream of the RNA coding sequence, and

ensures the proper location of transcription initiation.2) RNA coding sequence: the DNA sequence that is transcribed

into RNA.

3) terminator: a sequence downstream of the RNA coding

sequence and specifies where transcription will stop.



The prokaryote promoter sequences are generally found at -35

and -10 bp from the transcription start site.

-35 consensus sequence: 5’-TTGACA-3’ -10 consensus sequence: 5’-TATAAT-3’ (also called the Pribnow

box)

***Variation within these sequences results in variation in the binding ability of RNA polymerase and can affect the rate of

transcription.


http://slidepdf.com/reader/full/molecular-biology-lecture-4 11/26Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Initiation and elongation of the RNA transcript in E. coli.

a) RNA polymerase

(holoenzyme - core

RNA polymerase

plus sigma factor)

binds to -35 and -10

regions.

b) DNA molecule is

unwound by

approximately 17

bp in the -10 region.



c) After 8 or 9

ribonucleotides

have been

polymerized thesigma factor

disassociates with

the core enzyme.

d) Polymerization proceeds as RNA

polymerase

unwinds DNA

molecule.

e) Transcription is

terminated once a

termination

sequence is reached.



Eukaryotic promoters consist of a collection of conserved short

sequence elements located near the transcription start site:

Cis element DNA sequence Approx. Position

GC box GGGCGG -70 to -200

TATA box TATAAA -20 to -35

CAAT box CCAAT -80



Transcription Initiation in Eukaryotes Requires Many Proteins:

1) TATA box binding protein

-a subunit of TFIID-binds to TATA box of gene promoter

2) TFIID - causes a distortion in the DNA helix allowing the

recruitment of other transcription factors.

3) TFIIB - involved in RNA polymerase interactions

-start site recognition

4) TFIIH

- contains a DNA helicase to unwind DNA

-activates RNA polymerase by phosphorylation

5) TFIIE - involved in positioning RNA polymerase




Transcription initiation in eukaryotes:

1) The most common

eukaryote promoter

sequence is located at position -25 and

contains the

consensus sequence:

5’-TATAAAA-3’

(TATA BOX).

2) TBP/TFIID bind to

the promoter at the

TATA box (-25).

3) TFIIB binds to

promoter.



4) RNA polymerase, TFIIE, and

TFIIH are recruited to the

promoter and transcriptioninitiation site.

5) TFIIH unwinds the DNA.

6) TFIIH phosphorylates RNA

polymerase activating it for

transcription.

7) Most transcription factors arethen released from the basal

transcriptional machinery

prior to transcription

initiation.



The RNA pol II and the transcription

factors TFIID (TAFs and TBP), and

other transcription factors form the

basal transcriptional machinery.

The basal transcriptional machinery

is sufficient for only a low level oftranscription.

For increase levels of transcription or

tissue specific transcription, the binding of an activator and adaptor

molecule is required.



Transcription of DNA results in positive (+ve) supercoils in front

of the RNA polymerase and negative (-ve) supercoils behind the

RNA polymerase.

Transcribing DNA Overwound

(+ve supercoils)Underwound

(-ve supercoils)

Gyrase

Introduces -ve

supercoils

Topoisomerase

Relaxes -ve

supercoils



Termination of transcription can involve intrinsic termination

sequences found in the RNA transcript.

1) Palindromic regions that form hairpins varying in length from 7

to 20 bps.

2) The stem loop structure includes a G-C rich region and is

followed by a run of U residues.

G C

C

A

AA

G

C

C

C

U

A

A

G

U

UU

C

G

G

G

A

U

U

U U U U

A G

U

G

G

CC

G

U

A

G C

A

G

U

AG

G-C rich region in stem

Single-stranded U run



Termination of transcription can involve extrinsic factors such as

the rho protein.

1) Rho protein attaches to

recognition site on the RNA.

2) Rho moves along RNA following

RNA polymerase.

3) RNA polymerase pauses at

terminator and rho catches up.

4) Rho unwinds DNA-RNA hybrid

and pull RNA from the RNA polymerase.

5) Termination: RNA polymerase,

rho, and RNA are released.




General structure of mRNA found in bothprokaryotic and eukaryotic cells




In eukaryotes, the

initial produce of

transcription must

be processed and

modified to form

the mature mRNA.

1) Pre-mRNA is

spliced to remove

introns (non protein

coding regions).




Cap structure at the 5 end of a eukaryotic mRNA

2) The addition of a 7-methylguanosine to the 5’ end of the

mRNA.

This process is called 5’ cappingand is necessary for the

binding of the mRNA to the

ribosome which is essential for

the translation process.



3) The addition of

a poly-A tail tothe 3’ end of

the mRNA.

The poly-A tail is

necessary for

mRNA

stability.

Polyadenylation of mRNA



Fidelity of RNA polymerase

• RNA polymerase lacks the proof reading activity that is found

in DNA polymerase.

• the accuracy of transcription is much less than that of

replication.

• error rate is about 1/10,000 base pairs

• this level of error is tolerated in the cell because there many

transcripts from each gene and most will be error free

Molecular Biology Lecture 4

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