Lecture 21 - Molecular and Cell Biology

DNA: the genetic material
genetic information in the sequence of bases

How can this information be used in the cell?

First, the base sequence in DNA is used to synthesize RNA with complementary base sequence: Transcription

Second, the base sequence in RNA is used to synthesize polypeptides with "complementary" amino acid sequence: Translation

information flow from gene to protein, with base sequence in DNA determining amino acid sequence in protein. This concept is the:
Central Dogma of Molecular Biology

DNA-----transcription------> mRNA ------translation-----> protein


Transcription in E. coli (in prokaryotes):
Enzyme involved in transcription- DNA dependent RNA polymerase

RNA polymerase activity: some similarities, some differences to DNA polymerase activities

Similarities: 1) uses nucleoside 5'-triphosphates (NTPs) as precursors (Freifelder Fig. 9-1)
3) catalyzes phosphodiester bond between NTPs
4) uses DNA as template
7) base pairing determines correct base
8) growth of nucleic acid chain only in 5' to 3' direction
9) growing strand antiparallel to template strand

Differences:
1) uses ribonucleoside 5'-triphosphates instead of deoxyribonucleoside 5'-triphosphates (ATP, GTP, CTP, UTP)
2) can initiate the start of a new strand de novo (no primer necessary) (remember the RNA polymerase "primase" in replication)
3) a single strand of RNA is produced (only one strand of DNA used for RNA synthesis: the DNA template strand with complementary base sequence)
4) only short stretches of DNA are transcribed

E. coli RNA polymerase

Very large protein complex, consists of five subunits
2 identical alpha subunits and 1 each of beta, beta', and sigma

The sigma subunit dissociates from the enzyme easily - leaves shortly following initiation, critical for recognition of start of gene (more shortly).

holoenzyme - complete enzyme - all five subunits together
but basic polymerization reaction possible without sigma subunit -
core enzyme: 2 alpha, 1 beta and 1 beta'

RNA polymerase so large it is visible in EM (Freifelder Fig. 9-3) (Fig. 6.1)

Transcription process in 4 phases:
binding of RNA polymerase at specific site of DNA [PROMOTER] where gene starts
initiation
elongation
termination

Promoter
The region at start of gene which carries specific binding site for RNA polymerase is called promoter

The base where transcription starts is numbered +1, bases in front or "upstream" numbered with minus sign: -1, -2, -3 and so on)

A comparison of the base sequences upstream of many genes revealed similarities:
-10 sequence and -35 sequence

-10 consensus sequence: TATAAT (most promoters differ in one or two bases, the closer the sequence is to the consensus sequence the more efficiently the gene is transcribed)

There is also a consensus sequence also for -35 sequence [TTGACA]

(But: not all genes have the -10 and -35 sequence in promoter)

Phases of transcription

Specific binding of RNA polymerase:

• RNA polymerase can bind to double-stranded DNA nonspecifically with low affinity (low strength)
  
• The holoenzyme (with sigma subunit) binds specifically to both the -10 and -35 sequence with high affinity ---- resulting in a closed-promoter complex
  
• The holoenzyme unwinds about 15 bases in double-stranded DNA around the initiation site for transcription ---- resulting in an open-promoter complex

• Transcription starts by base pairing of two rNTPs that are joined (therefore, the very first ribonucleotide in a newly transcribed RNA retains the triphosphate)
  
• When approximately 10 rNTPs are jointed by phosphodiester bonds [resulting in the loss of 2 phosphate groups from each rNTP], the sigma subunit is released from the core enzyme
  

• The core enzyme leaves the promoter site
  
• The core enzyme travels along DNA template
  
• The DNA is unwound in front and is rewound behind so that approximately 17 base pairs are always unwound
  
• rNTPs are added to growing RNA via base pairing and phosphodiester bond formation

• The core enzyme encounters a termination signal
  
• RNA is released from DNA template and from the enzyme
  
• RNA polymerase dissociates from DNA

What is an inverted repeat? A sequence of several bases in double-stranded DNA that is repeated in an inverted fashion

Example:

• 5'.....GCCAGTGG........CCACTGGC....3'
  
• 3'.....CGGTCACC........GGTGACCG....5' (template strand)

transcribed RNA: 5'.......GCCAGUGG.......CCACUGGC.....3'

Consequently there are internal sequences in the transcribed RNA that are complementary and can therefore base pair to form a stem-loop structure

Some termination sequences lack the series of adenines which are transcribed in to URACILS on the RNA. The RNA in such situations needs assistance from a specific protein (termedRho) which is necessary for termination. In Rho dependent termination, the Rho protein forces the RNA to separate from the DNA template.

Comments or questions should be directed to Cheryl.Davis@wku.edu
Last Modified: April 1, 1998
All contents copyright (C) 1997.
Western Kentucky University.