Lecture no.

Lecture 3 RNA and Protein

 COMPONENTS OF RNA

Uracil instead of thymine

Alternating phosphates
and ribose sugars

2’ hydroxyl group
BASE PAIRING IN RNA DOUBLE HELICES

In addition to “normal” (Watson-Crick pairing), there are four non-

canonical pairs that can form by hydrogen bonding.
MORE THAN 50 MODIFIED BASES HAVE BEEN
FOUND IN RNA
RNA SECONDARY STRUCTURES

• Typical structures
formed by folded RNA
include non-canonical
base pairs (“wobble”),
hairpins, loops, bulges
and junctions.

• RNA can also form

double helices
• (A-RNA double helix)
SECONDARY AND TERTIARY tRNA STRUCTURES

Typical “cloverleaf” drawing versus a more realistic 3-D tRNA

structure. The “anticodon” is the functionally important bit, and
it sticks out to remain accessible.
 RNA PSEUDOKNOTS:
FIRST FOUND IN PLANT VIRUSES

Rietveld et al (1982) The tRNA-like structure at the 3′ terminus of

turnip yellow mosaic virus RNA…Nucleic Acids Res 10:1929–1946

RNA pseudoknots comprise functional domains within ribozymes,

self-splicing introns, ribonucleoprotein complexes, viral
genomes, and other biological systems.
An important piece of RNA architecture for the globular structure
capable of performing important biological functions.
Three examples of common A-minor motifs. A-MINOR MOTIF

A common “long range” interaction among

different parts of the RNA molecule. Good fit
between adenosine and the minor groove of a
RNA double helix.
 TETRALOOP MOTIF: BASE STACKING
INTERACTIONS STABILIZE THE STRUCTURE

A stem-loop that is particularly

stable due to base stacking.

Additional structures described in Allison text include the ribose

zipper motif and the kink-turn motif, both of which combine with
other structures to define a broad range of RNA structures.
PROTEIN-MEDIATED
RNA FOLDING

Unfolded RNA can become

misfolded, or can directly fold
into the proper structure.

Binding proteins (blue) can

stabilize the structure.

RNA “chaperones” can

prevent misfolding.
 RNA STRUCTURES CAN BE ASSAYED BY
CLEAVAGE OF THE BACKBONE

Hydroxyl radicals cleave at unprotected nucleotides. In a folded

structure, specific nucleotides are protected from cleavage.
 THE RNA WORLD:
EVOLUTION STARTING FROM RNA

RNA as info carrier RNA directs protein

and enzyme synthesis

RNA can perform many of the functions needed by a cell, including

encoding information, performing functions as catalysts (“ribozymes”),
regulating gene expression, binding proteins, etc.
 RIBOZYMES CAN CATALYZE REACTIONS –
LIKE A PROTEIN

The maturation of a tRNA is

catalyzed by RNase P (with protein
using RNA as a co-factor). The maturation of a tRNA
(production of 3’ and 5’ products)
requires protein (Rpp) and RNA.
STRUCTURAL SIMILARITIES OF RNA RIBOZYME
AND PROTEIN POLYMERASE

Both a self-splicing
intron and
bacteriophage T7
DNA polymerase
contain two metal
ions that coordinate
the substrate position
HAMMERHEAD RIBOZYMES: A FREQUENT
CATALYTIC MOTIF OF PLANT VIROIDS

Viroids = pathogenic RNAs.

The catalytic motifs of these small

ribozymes are all involved in
replication via “rolling circles”.
Long RNAs are self-cleaved into
monomers.

Small ribozymes of 40 to 154 nt

Predict RNA Structure from
websites
http://rna.tbi.univie.ac.at/
https://molbiol-tools.ca/RNA_analysis.htm
https://molbiol-tools.ca/RNA_analysis.htm
Special roles of RNA Structure
Yang et al., 2018, Front. Plant. Sci.
Wang et al., Nat. Chem. Biol. 17:755-766
Protein Structure
Protein Building Block:
Amino Acid
Resonance Structures in Peptide
Peptide Backbone
Levels of Structure in Protein
 Primary Structure

•Linear order of amino acids along with any

disulfide linkages between Cys is a protein’s
primary structure.
Secondary Structure: The a Helix
• The a-helix contains 3.6 amino
acid residues per turn with right-
handed spiral. One full turn is 5.4
Å long.
• The hydrogen on the amide
nitrogen forms a hydrogen bond
with the carbonyl oxygen of the
fourth residue towards the N -
terminus.
• Charged amino acids and proline
are less frequently in the packed
a-helix .
• The diameter of the a-helix is
about 12 Å long, similar to the
width of the major groove in DNA.
For this reason, a-helix are often
found in proteins that bind to
DNA.
Secondary Structure: b Sheet
• The structure of the sheet.
Hydrogen bonds are formed
between adjacent β strands.
• R groups extend out from
the β sheet, emphasizing the
pleated shape.
• (a) In an antiparallel β sheet,
the N- to C-terminal
orientation of the β strands
alternates (shown by the
arrowheads).
• (b) In a parallel β sheet, the
β strands align in the same
direction.
Linking Secondary Structures by b Turns

• secondary structural elements reverse themselves by

small and precise reverse turns, or loops such as b turn.
• b turn are often placed on the surface of the protein,
where the backbone can hydrogen-bound to water.
Supersecondary Structure (Motif): Building
blocks of Domains
• Particularly stable and
common
arrangements of
multiple secondary
structure elements
are called
supersecondary
structure or motif or
folds.
• Supersecondary
structure are the
building blocks
associated with
particular functions.
b Sheet Supersecondary Structure
Supersecondary Structure: a Helix & b Sheet
 Supersecondary Structure: The
Nucleotide-Binding Rossmann Fold

• This structure is commonly observed in ATP-binding or

GTP-binding proteins and is sometimes referred to as a
Rossman fold.
 Supersecondary Structure of a Helices

• The helix-turn-helix structure is commonly observed in

DNA binding motif of transcription factors.
• The four-helix bundle is formed by the interaction of four
α-helix.
Protein Domains: Independent Folding Units
within the Protein
• Compact structural regions of a
protein are referred to as domains
• Immunoglobulins provide an
example of 4 globular domains
• Domains may contain common
structural-functional motifs
– Zinc finger
– Hydrophobic pocket
 Tertiary Structure
• Total three-dimensional
shape of a polypeptide
is its tertiary structure
• A prominent aspect of
this structure is
interaction of the amino
acid side chains
• The globular form of a
polypeptide is a roughly
spherical structure
Tertiary Structure and Quaternary Structure
E. Coli DNA Polymerase
 Quaternary Structure

E Coli. CRP PCNA Human Hemoglobin

• Quaternary structure is the interaction of 2 or more
polypeptides.
• The quaternary structure range from a simple
homodimer to a large, multiprotein assembly such as
a ribosome.
Protein Folding: a Hierarchical Model
• Protein folding: A process in which the
polypeptide backbone folds to adopt a particular
conformation.
 Chaperon-assisted Protein Folding
• Chaperones are specialized proteins that bind improperly
folded polypeptides and facilitate correct folding pathways or
provide micro environments in which folding can occur.
Predict Protein Structure
https://molbiol-tools.ca/Protein_secondary_structure.htm
https://molbiol-tools.ca/Protein_secondary_structure.htm
https://www.alphafold.ebi.ac.uk/
PROTEIN FUNCTIONS
Proteins are highly
adaptable molecules and
vary greatly in size,
shape and function.

20% of cellular weight is

protein.
PROTEIN-LIGAND INTERACTIONS

• The bound molecule is called a ligand.

• A ligand binds at a site on the protein called

the binding site.

• Proteins exhibit conformational flexibility.

• Many protein ligand interactions require a

conformational change known as induced
fit.

• The subunits in a multisubunit protein often

exhibit cooperatively.
ENZYMES SERVE AS CATALYSTS TO LOWER ACTIVATION
ENERGIES

Enzymes catalyze
chemical reactions, and
can increase the reaction
rate by 108 to 1010 or even
higher.
REGULATION OF PROTEIN ACTIVITY
-Allosteric regulations

Negative control – binding Positive control – binding

blocks activity stimulates activity
 REGULATION OF PROTEIN ACTIVITY
-Phosphorylation regulation

A common cellular
mechanism for the
regulation of protein
activity. Phosphorylation
can activate or
deactivate proteins.
 ACTIVATION OF CDK BY CYCLIN BINDING AND
PHOSPHORYLATION

Cyclin-dependent kinases (CDKs) are protein kinases known for their role in regulating the cell cycle.
REGULATION OF PROTEIN FOLDING

Molecular chaperones Hsp70 and Hsp40

assist folding of newly synthesized proteins
 UBIQUITIN-MEDIATED
PROTEIN DEGRADATION

Proteins may be “tagged”

for selective destruction in
proteolytic complexes
called proteasomes

This tagging occurs by

covalent attachment of
ubiquitin, a small,
compact and highly
conserved protein.
 4.4 PROTEIN METHODS - OVERVIEW
The key to working with proteins are techniques that allow the biochemist to:

1) Separate distinct proteins

2) Differentiate or identify distinct proteins after separation
3) Concentrate and/or purify distinct proteins while retaining the function (e.g. the
correct folding cannot be lost)
4) Measure activity.
 PROTEIN METHODS - OVERVIEW
The key to working with proteins are techniques that allow the biochemist to:

1) Separate distinct proteins

2) Differentiate or identify distinct proteins after separation
3) Concentrate and/or purify distinct proteins while retaining the function (e.g. the
correct folding cannot be lost)
4) Measure activity.

Knowing the structure of a protein can tell you a great deal about the function and
how that protein interacts with other molecules (ligands or interacting proteins).
 PROTEIN METHODS - OVERVIEW
The key to working with proteins are techniques that allow the biochemist to:

1) Separate distinct proteins

2) Differentiate or identify distinct proteins after separation
3) Concentrate and/or purify distinct proteins while retaining the function (e.g. the
correct folding cannot be lost)
4) Measure activity.

Knowing the structure of a protein can tell you a great deal about the function and
how that protein interacts with other molecules (ligands or interacting proteins).

If the proteins are in complexes with other proteins, it is important to know:

5) What the other interacting proteins are.
6) The number of interacting proteins.
7) The relative ratios of each protein in the complex (the “stoichiometry”).
8) How they are assembled relative to one another.

And if the proteins are interacting with non-protein molecules, it’s important to know
what they are (e.g. small molecules, DNA, RNA, etc).
 PROTEIN SEPARATION - CENTRIFUGATION
Centrifugation is a very important
method that allows you to
separates molecules of different
sizes.
SEPARATION OF CELLULAR COMPONENTS

Proteins can be isolated from different cell

fractions, such as the plasma membrane, the
cytoplasm, the nucleus, etc. These fractions are
obtained by centrifugation. Different speeds
separate components of different molecular
weights.
 PROTEIN SEPARATION –
GEL ELECTROPHORESIS

Protein are separated on poly-

acrylamide gels.
 PROTEIN SEPARATION – 2-D
GEL ELECTROPHORESIS
Because of the different amino
acid side chains that are
exposed on the surface of the
protein, proteins have different
charges.

These charges can be used to

separate proteins by isoelectric
focusing in a pH gradient.

The combination of isoelectric

focusing and SDS gel
electrophoresis can separate
proteins in two dimensions,
hence the “2-D gel”.
An Approach to Studying DNA-Protein
Interaction: DNA Foot printing
 Studying DNA-Protein Interaction:
electrophoretic mobility shift assay (EMSA)
 What We have Covered Today
 RNA structure :
- Primary, secondary and tertiary structure
- Ribozymes
- Predict RNA structure
 Protein structure:
- Primary, secondary, tertiary and quaternary structure
-Protein folding
-Predict protein structure
-Protein function
-Work with protein

Read: most of these slides are from Cox’

Molecular Biology: Principles and Practice
(Chapter 4, 5 and 6 )